Human transferases

ABSTRACT

The invention provides three human transferases (HUTRAN) and polynucleotides which identify and encode HUTRAN. The invention also provides expression vectors, host cells, antibodies, agonists, and antagonists. The invention also provides methods for diagnosing, treating or preventing disorders associated with expression of HUTRAN.

FIELD OF THE INVENTION

This invention relates to nucleic acid and amino acid sequences of humantransferases and to the use of these sequences in the diagnosis,treatment, and prevention of autoimmune/inflammatory, neurological,reproductive, and gastrointestinal disorders and cancer.

BACKGROUND OF THE INVENTION

Transferases, enzymes that catalyze group transfer reactions, areclassified by the type of group transferred. One-carbon groups aretransferred; for example, methyltransferases transfer methyl groups fromS-adenosyl-methionine to substrates. Nitrogenous groups are transferred;for example, aminotransferases transfer amino groups. Other groupstransferred include aldehyde or ketone, acyl, glycosyl, alkyl and arylother than methyl, phosphorus-containing, sulfur-containing, andselenium-containing groups.

The enzyme glutamine-phenylpyruvate aminotransferase, also known asglutamine transaminase K (GTK), catalyzes several reactions with apyridoxal phosphate cofactor. GTK catalyzes the reversible conversion ofL-glutamine and phenylpyruvate to 2-oxoglutaramate and L-phenylalanine.L-methionine, L-histidine, and L-tyrosine can substitute for L-glutaminein this reaction. GTK also catalyzes the conversion of kynurenine tokynurenic acid. Kynurenic acid, a tryptophan metabolite, is anantagonist of the N-methyl-D-aspartate (NMDA) receptor in the brain andmay exert a neuromodulatory function. Alteration of the kynureninemetabolic pathway may be among the causative factors leading to severalneurological disorders. GTK also possesses cysteine conjugate β-lyaseactivity which is involved in the metabolism of halogenated xenobioticsconjugated to glutathione. GTK action on the cysteine conjugates ofxenobiotics yields metabolites that are nephrotoxic in rats andneurotoxic in humans. The neurotoxicity may be related to the kynurenineaminotransferase activity of GTK. GTK is expressed in kidney, liver, andbrain. Both cytosolic and mitochondrial forms exist. Human and rat GTKgenes have been isolated which encode proteins of 422 and 423 aminoacids respectively. Both human and rat GTKs contain a putative pyridoxalphosphate binding site. (ExPASy ENZYME: EC 2.6.1.64; Perry, S. J. et al.(1993) Mol. Pharmacol. 43:660-665; Perry, S. et al. (1995) FEBS Lett.360:277-280; and Alberati-Giani, D. et al. (1995) J. Neurochem.64:1448-1455.)

The enzyme kynurenine/α-aminoadipate aminotransferase (AadAT) catalyzestwo reactions with a pyridoxal phosphate cofactor. AadAT catalyzes thereversible conversion of α-aminoadipate and α-ketoglutarate toα-ketoadipate and L-glutamate. This conversion is involved in lysinemetabolism. AadAT also catalyzes the transamination of kynurenine acidto kynurenic acid. As described above, kynurenic acid is an NMDAreceptor antagonist. Both soluble and mitochondrial forms of AadAT havebeen purified. A soluble AadAT is expressed in rat kidney, liver, andbrain. The rat AadAT nucleotide gene encodes a protein of 425 aminoacids which contains a putative pyridoxal phosphate binding site.(Nakatani, Y. et al. (1970) Biochim. Biophys. Acta 198:219-228; Buchli,R. et al. (1995) J. Biol. Chem. 270:29330-29335.)

Protein-arginine methyltransferases catalyze the posttranslationalmethylation of arginine residues in proteins, resulting in the mono- anddimethylation of arginine on the guanidino group. Known substrates arehistones, heterogeneous nuclear ribonucleoproteins (hnRNPs), and myelinbasic protein. This otherwise unusual posttranslational modification iscommon in hnRNPs and may regulate their function. hnRNPs function in thenucleus in mRNA processing, splicing, and transport into the cytoplasm.Homologous protein-arginine methyltransferases that methylate hnRNPshave been cloned from yeast, rat, and man. These protein-argininemethyltransferases contain five sequence motifs, termed region I,post-region I, region II, region III, and post-region III, that may beinvolved in binding S-adenosyl-methionine. One human gene (HRMT1L1)encodes a 433 amino acid protein. The other human gene (HRMT1L2) may bealternatively spliced to yield three protein-argininemethyltransferases, of length 343, 347, and 361 amino acidsrespectively, with different amino termini. The protein encoded by thecloned rat protein-arginine methyltransferase gene (PRMT1) interactswith the TIS21 protein and the homologous BTG1 protein. Theintermediate-early TIS21 protein is the product of a gene induced bytreatment of cells with mitogens such as epidermal growth factor, andthe BTG1 protein is the product of a human gene located near achromosome translocation breakpoint associated with chronic lymphocyticleukemia. The HRMT 1L2 protein interacts with the cytoplasmic domain ofthe interferon receptor. This interaction suggests that proteinmethylation may be an important signaling mechanism for cytokinereceptors. (Lin, W.-J. et al. (1996) J. Biol. Chem. 271:15034-15044;Abramovich, C. et al. (1997) EMBO J. 16:260-266; and Scott, H. S. et al.(1998) Genomics 48:330-340.)

The discovery of new human transferases and the polynucleotides encodingthem satisfies a need in the art by providing new compositions which areuseful in the diagnosis, treatment, and prevention ofautoimmune/inflammatory, neurological, reproductive, andgastrointestinal disorders and cancer.

SUMMARY OF THE INVENTION

The invention features substantially purified polypeptides, humantransferases, referred to collectively as "HUTRAN" and individually as"HUTRAN-1", "HUTRAN-2", and "HUTRAN-3.". In one aspect, the inventionprovides a substantially purified polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,and SEQ ID NO:3 (SEQ ID NO:1-3) and fragments thereof.

The invention further provides a substantially purified variant havingat least 90% amino acid identity to the amino acid sequences of SEQ IDNO:1-3 or to fragments of any of these sequences. The invention alsoprovides an isolated and purified polynucleotide encoding thepolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-3 and fragments thereof. The invention alsoincludes an isolated and purified polynucleotide variant having at least70% polynucleotide sequence identity to the polynucleotide encoding thepolypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-3 and fragments thereof.

Additionally, the invention provides an isolated and purifiedpolynucleotide which hybridizes under stringent conditions to thepolynucleotide encoding the polypeptide comprising an amino acidsequence selected from the group consisting of SEQ ID NO:1-3 andfragments thereof, as well as an isolated and purified polynucleotidehaving a sequence which is complementary to the polynucleotide encodingthe polypeptide comprising the amino acid sequence selected from thegroup consisting of SEQ ID NO:1-3 and fragments thereof.

The invention also provides an isolated and purified polynucleotidecomprising a polynucleotide sequence selected from the group consistingof SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6 (SEQ ID NO:4-6), andfragments thereof. The invention further provides an isolated andpurified polynucleotide variant having at least 70% polynucleotidesequence identity to the polynucleotide sequence comprising apolynucleotide sequence selected from the group consisting of SEQ IDNO:4-6 and fragments thereof, as well as an isolated and purifiedpolynucleotide having a sequence which is complementary to thepolynucleotide comprising a polynucleotide sequence selected from thegroup consisting of SEQ ID NO:4-6 and fragments thereof.

The invention further provides an expression vector containing at leasta fragment of the polynucleotide encoding the polypeptide comprising anamino acid sequence selected from the group consisting of SEQ ID NO:1-3and fragments thereof. In another aspect, the expression vector iscontained within a host cell.

The invention also provides a method for producing a polypeptidecomprising the amino acid sequence selected from the group consisting ofSEQ ID NO:1-3 and fragments thereof, the method comprising the steps of:(a) culturing the host cell containing an expression vector containingat least a fragment of a polynucleotide encoding the polypeptide underconditions suitable for the expression of the polypeptide; and (b)recovering the polypeptide from the host cell culture.

The invention also provides a pharmaceutical composition comprising asubstantially purified polypeptide having the amino acid sequenceselected from the group consisting of SEQ ID NO:1-3 and fragmentsthereof in conjunction with a suitable pharmaceutical carrier.

The invention further includes a purified antibody which binds to apolypeptide comprising the amino acid sequence selected from the groupconsisting of SEQ ID NO:1-3 and fragments thereof, as well as a purifiedagonist and a purified antagonist to the polypeptide. The invention alsoprovides a method for treating or preventing an autoimmune/inflammatorydisorder, the method comprising administering to a subject in need ofsuch treatment an effective amount of an antagonist of the polypeptidehaving an amino acid sequence selected from the group consisting of SEQID NO:1-3 and fragments thereof.

The invention also provides a method for treating or preventing aneurological disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising a substantially purified polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:1-3and fragments thereof.

The invention also provides a method for treating or preventing areproductive disorder, the method comprising administering to a subjectin need of such treatment an effective amount of a pharmaceuticalcomposition comprising a substantially purified polypeptide having anamino acid sequence selected from the group consisting of SEQ ID NO:1-3and fragments thereof.

The invention also provides a method for treating or preventing agastrointestinal disorder, the method comprising administering to asubject in need of such treatment an effective amount of apharmaceutical composition comprising a substantially purifiedpolypeptide having an amino acid sequence selected from the groupconsisting of SEQ ID NO:1-3 and fragments thereof.

The invention also provides a method for treating or preventing acancer, the method comprising administering to a subject in need of suchtreatment an effective amount of an antagonist of the polypeptide havingan amino acid sequence selected from the group consisting of SEQ IDNO:1-3 and fragments thereof.

The invention also provides a method for detecting a polynucleotideencoding the polypeptide comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-3 and fragments thereof in abiological sample containing nucleic acids, the method comprising thesteps of: (a) hybridizing the complement of the polynucleotide sequenceencoding the polypeptide comprising the amino acid sequence selectedfrom the group consisting of SEQ ID NO:1-3 and fragments thereof to atleast one of the nucleic acids of the biological sample, thereby forminga hybridization complex; and (b) detecting the hybridization complex,wherein the presence of the hybridization complex correlates with thepresence of a polynucleotide encoding the polypeptide in the biologicalsample. In one aspect, the method further comprises amplifying thepolynucleotide prior to the hybridizing step.

BRIEF DESCRIPTION OF THE FIGURES AND TABLES

FIGS. 1A and 1B show the amino acid sequence alignment between HUTRAN-1(1815528; SEQ ID NO:1) and human glutamine-phenylpyruvateaminotransferase (GI 758591; SEQ ID NO:30), produced using themultisequence alignment program of LASERGENE™ software (DNASTAR Inc,Madison Wis.).

FIGS. 2A and 2B show the amino acid sequence alignment between HUTRAN-2(2150892; SEQ ID NO:2) and rat kynurenine/α-aminoadipateaminotransferase (GI 1050752; SEQ ID NO:31).

FIGS. 3A, 3B, and 3C show the amino acid sequence alignment betweenHUTRAN-3 (2525071; SEQ ID NO:3) and human arginine methyltransferase (GI1808648; SEQ ID NO:32).

In Table 1, columns 1 and 2 show the sequence identification numbers(SEQ ID NO:) of the amino acid and nucleic acid sequence, respectively.Column 3 shows the Clone ID of the Incyte Clone in which nucleic acidsencoding each HUTRAN were first identified, and column 4, the cDNAlibrary of this clone. Column 5 shows the Incyte clones (and libraries)and shotgun sequences useful as fragments in hybridization technologies,and which are part of the consensus nucleotide sequence of each HUTRAN.

Table 2 shows various properties of the polypeptides of the invention:column 1 references the SEQ ID NO; column 2 shows the number of aminoacid residues; column 3, potential phosphorylation sites; column 4,potential glycosylation sites; column 5, signature sequences associatedwith known proteins; column 6, the identity of the protein; and column7, analytical methods used to identify the protein through sequencehomologies, protein motifs, and protein signatures.

Table 3 shows the tissue expression of each nucleic acid sequence bynorthern analysis, diseases or conditions associated with this tissueexpression, and the vector into which each cDNA was cloned.

Table 4 describes the tissues used in cDNA library construction.

Table 5 describes the programs, algorithms, databases, and qualifyingscores used to analyze HUTRAN. The first column of Table 5 shows thetool, program, or algorithm; the second column, the database; the thirdcolumn, a brief description; and the fourth column (where applicable),scores for determining the strength of a match between two sequences(the higher the value, the more homologous).

DESCRIPTION OF THE INVENTION

Before the present proteins, nucleotide sequences, and methods aredescribed, it is understood that this invention is not limited to theparticular methodology, protocols, cell lines, vectors, and reagentsdescribed, as these may vary. It is also to be understood that theterminology used herein is for the purpose of describing particularembodiments only, and is not intended to limit the scope of the presentinvention which will be limited only by the appended claims.

It must be noted that as used herein and in the appended claims, thesingular forms "a," "an," and "the" include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to "ahost cell" includes a plurality of such host cells, and a reference to"an antibody" is a reference to one or more antibodies and equivalentsthereof known to those skilled in the art, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the preferred methods,devices, and materials are now described. All publications mentionedherein are cited for the purpose of describing and disclosing the celllines, vectors, and methodologies which are reported in the publicationsand which might be used in connection with the invention. Nothing hereinis to be construed as an admission that the invention is not entitled toantedate such disclosure by virtue of prior invention.

Definitions

"HUTRAN," as used herein, refers to the amino acid sequences ofsubstantially purified HUTRAN obtained from any species, particularly amammalian species, including bovine, ovine, porcine, murine, equine, andpreferably the human species, from any source, whether natural,synthetic, semi-synthetic, or recombinant.

The term "agonist," as used herein, refers to a molecule which, whenbound to HUTRAN, increases or prolongs the duration of the effect ofHUTRAN. Agonists may include proteins, nucleic acids, carbohydrates, orany other molecules which bind to and modulate the effect of HUTRAN.

An "allelic variant," as this term is used herein, is an alternativeform of the gene encoding HUTRAN. Allelic variants may result from atleast one mutation in the nucleic acid sequence and may result inaltered mRNAs or in polypeptides whose structure or function may or maynot be altered. Any given natural or recombinant gene may have none,one, or many allelic forms. Common mutational changes which give rise toallelic variants are generally ascribed to natural deletions, additions,or substitutions of nucleotides. Each of these types of changes mayoccur alone, or in combination with the others, one or more times in agiven sequence.

"Altered" nucleic acid sequences encoding HUTRAN, as described herein,include those sequences with deletions, insertions, or substitutions ofdifferent nucleotides, resulting in a polynucleotide the same as HUTRANor a polypeptide with at least one functional characteristic of HUTRAN.Included within this definition are polymorphisms which may or may notbe readily detectable using a particular oligonucleotide probe of thepolynucleotide encoding HUTRAN, and improper or unexpected hybridizationto allelic variants, with a locus other than the normal chromosomallocus for the polynucleotide sequence encoding HUTRAN. The encodedprotein may also be "altered," and may contain deletions, insertions, orsubstitutions of amino acid residues which produce a silent change andresult in a functionally equivalent HUTRAN. Deliberate amino acidsubstitutions may be made on the basis of similarity in polarity,charge, solubility, hydrophobicity, hydrophilicity, and/or theamphipathic nature of the residues, as long as the biological orimmunological activity of HUTRAN is retained. For example, negativelycharged amino acids may include aspartic acid and glutamic acid,positively charged amino acids may include lysine and arginine, andamino acids with uncharged polar head groups having similarhydrophilicity values may include leucine, isoleucine, and valine;glycine and alanine; asparagine and glutamine; serine and threonine; andphenylalanine and tyrosine.

The terms "amino acid" or "amino acid sequence," as used herein, referto an oligopeptide, peptide, polypeptide, or protein sequence, or afragment of any of these, and to naturally occurring or syntheticmolecules. In this context, "fragments," "immunogenic fragments," or"antigenic fragments" refer to fragments of HUTRAN which are preferablyat least 5 to about 15 amino acids in length, most preferably at least14 amino acids, and which retain some biological activity orimmunological activity of HUTRAN. Where "amino acid sequence" is recitedherein to refer to an amino acid sequence of a naturally occurringprotein molecule, "amino acid sequence" and like terms are not meant tolimit the amino acid sequence to the complete native amino acid sequenceassociated with the recited protein molecule.

"Amplification," as used herein, relates to the production of additionalcopies of a nucleic acid sequence. Amplification is generally carriedout using polymerase chain reaction (PCR) technologies well known in theart. (See, e.g., Dieffenbach, C. W. and G. S. Dveksler (1995) PCRPrimer, a Laboratory Manual, Cold Spring Harbor Press, Plainview, N.Y.,pp.1-5.)

The term "antagonist," as it is used herein, refers to a molecule which,when bound to HUTRAN, decreases the amount or the duration of the effectof the biological or immunological activity of HUTRAN. Antagonists mayinclude proteins, nucleic acids, carbohydrates, antibodies, or any othermolecules which decrease the effect of HUTRAN.

As used herein, the term "antibody" refers to intact molecules as wellas to fragments thereof, such as Fab, F(ab')₂, and Fv fragments, whichare capable of binding the epitopic determinant. Antibodies that bindHUTRAN polypeptides can be prepared using intact polypeptides or usingfragments containing small peptides of interest as the immunizingantigen. The polypeptide or oligopeptide used to immunize an animal(e.g., a mouse, a rat, or a rabbit) can be derived from the translationof RNA, or synthesized chemically, and can be conjugated to a carrierprotein if desired. Commonly used carriers that are chemically coupledto peptides include bovine serum albumin, thyroglobulin, and keyholelimpet hemocyanin (KLH). The coupled peptide is then used to immunizethe animal.

The term "antigenic determinant," as used herein, refers to thatfragment of a molecule (i.e., an epitope) that makes contact with aparticular antibody. When a protein or a fragment of a protein is usedto immunize a host animal, numerous regions of the protein may inducethe production of antibodies which bind specifically to antigenicdeterminants (given regions or three-dimensional structures on theprotein). An antigenic determinant may compete with the intact antigen(i.e., the immunogen used to elicit the immune response) for binding toan antibody.

The term "antisense," as used herein, refers to any compositioncontaining a nucleic acid sequence which is complementary to the "sense"strand of a specific nucleic acid sequence. Antisense molecules may beproduced by any method including synthesis or transcription. Onceintroduced into a cell, the complementary nucleotides combine withnatural sequences produced by the cell to form duplexes and to blockeither transcription or translation. The designation "negative" canrefer to the antisense strand, and the designation "positive" can referto the sense strand.

As used herein, the term "biologically active," refers to a proteinhaving structural, regulatory, or biochemical functions of a naturallyoccurring molecule. Likewise, "immunologically active" refers to thecapability of the natural, recombinant, or synthetic HUTRAN, or of anyoligopeptide thereof, to induce a specific immune response inappropriate animals or cells and to bind with specific antibodies.

The terms "complementary" or "complementarity," as used herein, refer tothe natural binding of polynucleotides by base pairing. For example, thesequence "5' A-G-T 3'" binds to the complementary sequence "3'T-C-A 5'."Complementarity between two single-stranded molecules may be "partial,"such that only some of the nucleic acids bind, or it may be "complete,"such that total complementarity exists between the single strandedmolecules. The degree of complementarity between nucleic acid strandshas significant effects on the efficiency and strength of thehybridization between the nucleic acid strands. This is of particularimportance in amplification reactions, which depend upon binding betweennucleic acids strands, and in the design and use of peptide nucleic acid(PNA) molecules.

A "composition comprising a given polynucleotide sequence" or a"composition comprising a given amino acid sequence," as these terms areused herein, refer broadly to any composition containing the givenpolynucleotide or amino acid sequence. The composition may comprise adry formulation or an aqueous solution. Compositions comprisingpolynucleotide sequences encoding HUTRAN or fragments of HUTRAN may beemployed as hybridization probes. The probes may be stored infreeze-dried form and may be associated with a stabilizing agent such asa carbohydrate. In hybridizations, the probe may be deployed in anaqueous solution containing salts, e.g., NaCl, detergents, e.g.,sodiumdodecyl sulfate (SDS), and other components, e.g., Denhardt's solution,dry milk, salmon sperm DNA, etc.

"Consensus sequence," as used herein, refers to a nucleic acid sequencewhich has been resequenced to resolve uncalled bases, extended usingXL-PCR™ (Perkin Elmer, Norwalk, Conn.) in the 5' and/or the 3'direction, and resequenced, or which has been assembled from theoverlapping sequences of more than one Incyte Clone using a computerprogram for fragment assembly, such as the GELVIEW™ fragment assemblysystem (GCG, Madison, Wis.). Some sequences have been both extended andassembled to produce the consensus sequence.

As used herein, the term "correlates with expression of apolynucleotide" indicates that the detection of the presence of nucleicacids, the same or related to a nucleic acid sequence encoding HUTRAN,by Northern analysis is indicative of the presence of nucleic acidsencoding HUTRAN in a sample, and thereby correlates with expression ofthe transcript from the polynucleotide encoding HUTRAN.

A "deletion," as the term is used herein, refers to a change in theamino acid or nucleotide sequence that results in the absence of one ormore amino acid residues or nucleotides.

The term "derivative," as used herein, refers to the chemicalmodification of a polypeptide sequence, or a polynucleotide sequence.Chemical modifications of a polynucleotide sequence can include, forexample, replacement of hydrogen by an alkyl, acyl, or amino group. Aderivative polynucleotide encodes a polypeptide which retains at leastone biological or immunological function of the natural molecule. Aderivative polypeptide is one modified by glycosylation, pegylation, orany similar process that retains at least one biological orimmunological function of the polypeptide from which it was derived.

The term "similarity," as used herein, refers to a degree ofcomplementarity. There may be partial similarity or complete similarity.The word "identity" may substitute for the word "similarity." Apartially complementary sequence that at least partially inhibits anidentical sequence from hybridizing to a target nucleic acid is referredto as "substantially similar." The inhibition of hybridization of thecompletely complementary sequence to the target sequence may be examinedusing a hybridization assay (Southern or Northern blot, solutionhybridization, and the like) under conditions of reduced stringency. Asubstantially similar sequence or hybridization probe will compete forand inhibit the binding of a completely similar (identical) sequence tothe target sequence under conditions of reduced stringency. This is notto say that conditions of reduced stringency are such that non-specificbinding is permitted, as reduced stringency conditions require that thebinding of two sequences to one another be a specific (i.e., aselective) interaction. The absence of non-specific binding may betested by the use of a second target sequence which lacks even a partialdegree of complementarity (e.g., less than about 30% similarity oridentity). In the absence of non-specific binding, the substantiallysimilar sequence or probe will not hybridize to the secondnon-complementary target sequence.

The phrases "percent identity" or "% identity" refer to the percentageof sequence similarity found in a comparison of two or more amino acidor nucleic acid sequences. Percent identity can be determinedelectronically, e.g., by using the MegAlign™ program (DNASTAR, Inc.,Madison Wis.). The MegAlign™ program can create alignments between twoor more sequences according to different methods, e.g., the clustalmethod. (See, e.g., Higgins, D. G. and P. M. Sharp (1988) Gene73:237-244.) The clustal algorithm groups sequences into clusters byexamining the distances between all pairs. The clusters are alignedpairwise and then in groups. The percentage similarity between two aminoacid sequences, e.g., sequence A and sequence B, is calculated bydividing the length of sequence A, minus the number of gap residues insequence A, minus the number of gap residues in sequence B, into the sumof the residue matches between sequence A and sequence B, times onehundred. Gaps of low or of no similarity between the two amino acidsequences are not included in determining percentage similarity. Percentidentity between nucleic acid sequences can also be counted orcalculated by other methods known in the art, e.g., the Jotun Heinmethod. (See, e.g., Hein, J. (1990) Methods Enzymol. 183:626-645.)Identity between sequences can also be determined by other methods knownin the art, e.g., by varying hybridization conditions.

"Human artificial chromosomes" (HACs), as described herein, are linearmicrochromosomes which may contain DNA sequences of about 6 kb to 10 Mbin size, and which contain all of the elements required for stablemitotic chromosome segregation and maintenance. (See, e.g., Harrington,J. J. et al. (1997) Nat Genet. 15:345-355.)

The term "humanized antibody," as used herein, refers to antibodymolecules in which the amino acid sequence in the non-antigen bindingregions has been altered so that the antibody more closely resembles ahuman antibody, and still retains its original binding ability.

"Hybridization," as the term is used herein, refers to any process bywhich a strand of nucleic acid binds with a complementary strand throughbase pairing.

As used herein, the term "hybridization complex" refers to a complexformed between two nucleic acid sequences by virtue of the formation ofhydrogen bonds between complementary bases. A hybridization complex maybe formed in solution (e.g., C₀ t or R₀ t analysis) or formed betweenone nucleic acid sequence present in solution and another nucleic acidsequence immobilized on a solid support (e.g., paper, membranes,filters, chips, pins or glass slides, or any other appropriate substrateto which cells or their nucleic acids have been fixed).

The words "insertion" or "addition," as used herein, refer to changes inan amino acid or nucleotide sequence resulting in the addition of one ormore amino acid residues or nucleotides, respectively, to the sequencefound in the naturally occurring molecule.

"Immune response" can refer to conditions associated with inflammation,trauma, immune disorders, or infectious or genetic disease, etc. Theseconditions can be characterized by expression of various factors, e.g.,cytokines, chemokines, and other signaling molecules, which may affectcellular and systemic defense systems.

The term "microarray," as used herein, refers to an arrangement ofdistinct polynucleotides arrayed on a substrate, e.g., paper, nylon orany other type of membrane, filter, chip, glass slide, or any othersuitable solid support.

The terms "element" or "array element" as used herein in a microarraycontext, refer to hybridizable polynucleotides arranged on the surfaceof a substrate.

The term "modulate," as it appears herein, refers to a change in theactivity of HUTRAN. For example, modulation may cause an increase or adecrease in protein activity, binding characteristics, or any otherbiological, functional, or immunological properties of HUTRAN.

The phrases "nucleic acid" or "nucleic acid sequence," as used herein,refer to a nucleotide, oligonucleotide, polynucleotide, or any fragmentthereof. These phrases also refer to DNA or RNA of genomic or syntheticorigin which may be single-stranded or double-stranded and may representthe sense or the antisense strand, to peptide nucleic acid (PNA), or toany DNA-like or RNA-like material. In this context, "fragments" refersto those nucleic acid sequences which, when translated, would producepolypeptides retaining some functional characteristic, e.g.,antigenicity, or structural domain characteristic, e.g., ATP-bindingsite, of the full-length polypeptide.

The terms "operably associated" or "operably linked," as used herein,refer to functionally related nucleic acid sequences. A promoter isoperably associated or operably linked with a coding sequence if thepromoter controls the translation of the encoded polypeptide. Whileoperably associated or operably linked nucleic acid sequences can becontiguous and in the same reading frame, certain genetic elements,e.g., repressor genes, are not contiguously linked to the sequenceencoding the polypeptide but still bind to operator sequences thatcontrol expression of the polypeptide.

The term "oligonucleotide," as used herein, refers to a nucleic acidsequence of at least about 6 nucleotides to 60 nucleotides, preferablyabout 15 to 30 nucleotides, and most preferably about 20 to 25nucleotides, which can be used in PCR amplification or in ahybridization assay or microarray. As used herein, the term"oligonucleotide" is substantially equivalent to the terms "amplimer,""primer," "oligomer," and "probe," as these terms are commonly definedin the art.

"Peptide nucleic acid" (PNA), as used herein, refers to an antisensemolecule or anti-gene agent which comprises an oligonucleotide of atleast about 5 nucleotides in length linked to a peptide backbone ofamino acid residues ending in lysine. The terminal lysine conferssolubility to the composition. PNAs preferentially bind complementarysingle stranded DNA or RNA and stop transcript elongation, and may bepegylated to extend their lifespan in the cell. (See, e.g., Nielsen, P.E. et al. (1993) Anticancer Drug Des. 8:53-63.)

The term "sample," as used herein, is used in its broadest sense. Abiological sample suspected of containing nucleic acids encoding HUTRAN,or fragments thereof, or HUTRAN itself, may comprise a bodily fluid; anextract from a cell, chromosome, organelle, or membrane isolated from acell; a cell; genomic DNA, RNA, or cDNA, in solution or bound to a solidsupport; a tissue; a tissue print; etc.

As used herein, the terms "specific binding" or "specifically binding"refer to that interaction between a protein or peptide and an agonist,an antibody, or an antagonist. The interaction is dependent upon thepresence of a particular structure of the protein, e.g., the antigenicdeterminant or epitope, recognized by the binding molecule. For example,if an antibody is specific for epitope "A," the presence of apolypeptide containing the epitope A, or the presence of free unlabeledA, in a reaction containing free labeled A and the antibody will reducethe amount of labeled A that binds to the antibody.

As used herein, the term "stringent conditions" refers to conditionswhich permit hybridization between polynucleotides and the claimedpolynucleotides. Stringent conditions can be defined by saltconcentration, the concentration of organic solvent, e.g., formamide,temperature, and other conditions well known in the art. In particular,stringency can be increased by reducing the concentration of salt,increasing the concentration of formamide, or raising the hybridizationtemperature.

The term "substantially purified," as used herein, refers to nucleicacid or amino acid sequences that are removed from their naturalenvironment and are isolated or separated, and are at least about 60%free, preferably about 75% free, and most preferably about 90% free fromother components with which they are naturally associated.

A "substitution," as used herein, refers to the replacement of one ormore amino acids or nucleotides by different amino acids or nucleotides,respectively.

"Transformation," as defined herein, describes a process by whichexogenous DNA enters and changes a recipient cell. Transformation mayoccur under natural or artificial conditions according to variousmethods well known in the art, and may rely on any known method for theinsertion of foreign nucleic acid sequences into a prokaryotic oreukaryotic host cell. The method for transformation is selected based onthe type of host cell being transformed and may include, but is notlimited to, viral infection, electroporation, heat shock, lipofection,and particle bombardment. The term "transformed" cells includes stablytransformed cells in which the inserted DNA is capable of replicationeither as an autonomously replicating plasmid or as part of the hostchromosome, as well as transiently transformed cells which express theinserted DNA or RNA for limited periods of time.

A "variant" of HUTRAN polypeptides, as used herein, refers to an aminoacid sequence that is altered by one or more amino acid residues. Thevariant may have "conservative" changes, wherein a substituted aminoacid has similar structural or chemical properties (e.g., replacement ofleucine with isoleucine). More rarely, a variant may have"nonconservative" changes (e.g., replacement of glycine withtryptophan). Analogous minor variations may also include amino aciddeletions or insertions, or both. Guidance in determining which aminoacid residues may be substituted, inserted, or deleted withoutabolishing biological or immunological activity may be found usingcomputer programs well known in the art, for example, LASERGENE™software.

The term "variant," when used in the context of a polynucleotidesequence, may encompass a polynucleotide sequence related to HUTRAN.This definition may also include, for example, "allelic" (as definedabove), "splice," "species," or "polymorphic" variants. A splice variantmay have significant identity to a reference molecule, but willgenerally have a greater or lesser number of polynucleotides due toalternate splicing of exons during mRNA processing. The correspondingpolypeptide may possess additional functional domains or an absence ofdomains. Species variants are polynucleotide sequences that vary fromone species to another. The resulting polypeptides generally will havesignificant amino acid identity relative to each other. A polymorphicvariant is a variation in the polynucleotide sequence of a particulargene between individuals of a given species. Polymorphic variants alsomay encompass "single nucleotide polymorphisms" (SNPs) in which thepolynucleotide sequence varies by one base. The presence of SNPs may beindicative of, for example, a certain population, a disease state, or apropensity for a disease state.

The Invention

The invention is based on the discovery of three new human transferases(HUTRAN), the polynucleotides encoding HUTRAN, and the use of thesecompositions for the diagnosis, treatment, or prevention ofautoimmune/inflammatory, neurological, reproductive, andgastrointestinal disorders and cancer. Table 1 summarizes the sequenceidentification numbers, identifying clone numbers, and libraries ofHUTRAN.

As shown in Table 2, each HUTRAN has been characterized with regard toits chemical and structural similarity with transferase molecules. Asshown in FIGS. 1A and 1B, HUTRAN-1 and human glutamine-phenylpyruvateaminotransferase (GI 758591; SEQ ID NO:30) share 49% identity. As shownin FIGS. 2A and 2B, HUTRAN-2 and rat kynurenine/α-aminoadipateaminotransferase (GI 1050752; SEQ ID NO:31) share 71% identity. As shownin FIGS. 3A, 3B, and 3C, HUTRAN-3 and human arginine methyltransferase(GI 1808648; SEQ ID NO:32) share 27% identity.

In Table 3, northern analysis shows the expression of HUTRAN sequencesin various libraries, of which at least 42% are immortalized orcancerous, at least 18% are in fetal or proliferating tissue, at least9% involve trauma, and at least 14% involve immune response. Ofparticular note is the expression of HUTRAN in male and femalereproductive, nervous, and gastrointestinal tissues.

A preferred HUTRAN variant is one which has at least about 80%, morepreferably at least about 90%, and most preferably at least about 95%amino acid sequence identity to the HUTRAN amino acid sequence, andwhich contains at least one functional or structural characteristic ofHUTRAN.

The invention also encompasses polynucleotides which encode HUTRAN. In aparticular embodiment, the invention encompasses a polynucleotidesequence comprising the sequence of SEQ ID NO:4, which encodes a HUTRAN.In a further embodiment, the invention encompasses the polynucleotidesequence comprising the sequence of SEQ ID NO:5. In a furtherembodiment, the invention encompasses the polynucleotide sequencecomprising the sequence of SEQ ID NO:6.

The invention also encompasses a variant of a polynucleotide sequenceencoding HUTRAN. In particular, such a variant polynucleotide sequencewill have at least about 70%, more preferably at least about 85%, andmost preferably at least about 95% polynucleotide sequence identity tothe polynucleotide sequence encoding HUTRAN. Any one of thepolynucleotide variants described above can encode an amino acidsequence which contains at least one functional or structuralcharacteristic of HUTRAN.

It will be appreciated by those skilled in the art that as a result ofthe degeneracy of the genetic code, a multitude of polynucleotidesequences encoding HUTRAN, some bearing minimal similarity to thepolynucleotide sequences of any known and naturally occurring gene, maybe produced. Thus, the invention contemplates each and every possiblevariation of polynucleotide sequence that could be made by selectingcombinations based on possible codon choices. These combinations aremade in accordance with the standard triplet genetic code as applied tothe polynucleotide sequence of naturally occurring HUTRAN, and all suchvariations are to be considered as being specifically disclosed.

Although nucleotide sequences which encode HUTRAN and its variants arepreferably capable of hybridizing to the nucleotide sequence of thenaturally occurring HUTRAN under appropriately selected conditions ofstringency, it may be advantageous to produce nucleotide sequencesencoding HUTRAN or its derivatives possessing a substantially differentcodon usage, e.g., inclusion of non-naturally occurring codons. Codonsmay be selected to increase the rate at which expression of the peptideoccurs in a particular prokaryotic or eukaryotic host in accordance withthe frequency with which particular codons are utilized by the host.Other reasons for substantially altering the nucleotide sequenceencoding HUTRAN and its derivatives without altering the encoded aminoacid sequences include the production of RNA transcripts having moredesirable properties, such as a greater half-life, than transcriptsproduced from the naturally occurring sequence.

The invention also encompasses production of DNA sequences which encodeHUTRAN and HUTRAN derivatives, or fragments thereof, entirely bysynthetic chemistry. After production, the synthetic sequence may beinserted into any of the many available expression vectors and cellsystems using reagents well known in the art. Moreover, syntheticchemistry may be used to introduce mutations into a sequence encodingHUTRAN or any fragment thereof.

Also encompassed by the invention are polynucleotide sequences that arecapable of hybridizing to the claimed polynucleotide sequences, and, inparticular, to those shown in SEQ ID NO:4-6 and fragments thereof undervarious conditions of stringency. (See, e.g., Wahl, G. M. and S. L.Berger (1987) Methods Enzymol. 152:399-407; Kimmel, A. R. (1987) MethodsEnzymol. 152:507-511.) For example, stringent salt concentration willordinarily be less than about 750 mM NaCl and 75 mM trisodium citrate,preferably less than about 500 mM NaCl and 50 mM trisodium citrate, andmost preferably less than about 250 mM NaCl and 25 mM trisodium citrate.Low stringency hybridization can be obtained in the absence of organicsolvent, e.g., formamide, while high stringency hybridization can beobtained in the presence of at least about 35% formamide, and mostpreferably at least about 50% formamide. Stringent temperatureconditions will ordinarily include temperatures of at least about 30°C., more preferably of at least about 37° C., and most preferably of atleast about 42° C. Varying additional parameters, such as hybridizationtime, the concentration of detergent, e.g., sodium dodecyl sulfate(SDS), and the inclusion or exclusion of carrier DNA, are well known tothose skilled in the art. Various levels of stringency are accomplishedby combining these various conditions as needed. In a preferredembodiment, hybridization will occur at 30° C. in 750 mM NaCl, 75 mMtrisodium citrate, and 1% SDS. In a more preferred embodiment,hybridization will occur at 37+ C. in 500 mM NaCl, 50 mM trisodiumcitrate, 1% SDS, 35% formamide, and 100 μg/ml denatured salmon sperm DNA(ssDNA). In a most preferred embodiment, hybridization will occur at 42°C. in 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50% formamide, and200 μg/ml ssDNA. Useful variations on these conditions will be readilyapparent to those skilled in the art.

The washing steps which follow hybridization can also vary instringency. Wash stringency conditions can be defined by saltconcentration and by temperature. As above, wash stringency can beincreased by decreasing salt concentration or by increasing temperature.For example, stringent salt concentration for the wash steps willpreferably be less than about 30 mM NaCl and 3 mM trisodium citrate, andmost preferably less than about 15 mM NaCl and 1.5 mM trisodium citrate.Stringent temperature conditions for the wash steps will ordinarilyinclude temperature of at least about 25° C., more preferably of atleast about 42° C., and most preferably of at least about 68° C. In apreferred embodiment, wash steps will occur at 25° C. in 30 mM NaCl, 3mM trisodium citrate, and 0.1% SDS. In a more preferred embodiment, washsteps will occur at 42° C. in 15 mM NaCl, 1.5 mM trisodium citrate, and0.1% SDS. In a most preferred embodiment, wash steps will occur at 68°C. in 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS. Additionalvariations on these conditions will be readily apparent to those skilledin the art.

Methods for DNA sequencing and analysis are well known in the art. Themethods may employ such enzymes as the Klenow fragment of DNA polymeraseI, SEQUENASE® (Amersham Pharmacia Biotech Ltd., Uppsala, Sweden), Taqpolymerase (The Perkin-Elmer Corp., Norwalk, Conn.), thermostable T7polymerase (Amersham Pharmacia Biotech Ltd., Uppsala, Sweden), orcombinations of polymerases and proofreading exonucleases, such as thosefound in the ELONGASE™ amplification system (Life Technologies, Inc.,Gaithersburg, Md.). Preferably, sequence preparation is automated withmachines, e.g., the ABI CATALYST™ 800 (Perkin Elmer) or MICROLAB® 2200(Hamilton Co., Reno, Nev.) sequence preparation machines, in combinationwith thermal cyclers. Sequencing can also be automated, such as byABIPRISM™ 373 or 377 DNA sequencers (The Perkin-Elmer Corp., Norwalk,Conn.) or the MEGABACE™ 1000 capillary electrophoresis system (MolecularDynamics, Inc., Sunnyvale, Calif.). Sequences can be analyzed usingcomputer programs and algorithms well known in the art. (See, e.g.,Ausubel, supra, unit 7.7; and Meyers, R. A. (1995) Molecular Biology andBiotechnology, Wiley VCH, Inc, New York, N.Y.).

The nucleic acid sequences encoding HUTRAN may be extended utilizing apartial nucleotide sequence and employing various PCR-based methodsknown in the art to detect upstream sequences, such as promoters andregulatory elements. For example, one method which may be employed,restriction-site PCR, uses universal and nested primers to amplifyunknown sequence from genomic DNA within a cloning vector. (See, e.g.,Sarkar, G. (1993) PCR Methods Applic. 2:318-322.) Another method,inverse PCR, uses primers that extend in divergent directions to amplifyunknown sequence from a circularized template. The template is derivedfrom restriction fragments comprising a known genomic locus andsurrounding sequences. (See, e.g., Triglia, T. et al. (1988) NucleicAcids Res. 16:8186.) A third method, capture PCR, involves PCRamplification of DNA fragments adjacent to known sequences in human andyeast artificial chromosome DNA. (See, e.g., Lagerstrom, M. et al.(1991) PCR Methods Applic. 1:111-119.) In this method, multiplerestriction enzyme digestions and ligations may be used to insert anengineered double-stranded sequence into a region of unknown sequencebefore performing PCR. Other methods which may be used to retrieveunknown sequences are known in the art. (See, e.g., Parker, J. D. et al.(1991) Nucleic Acids Res. 19:3055-306). Additionally, one may use PCR,nested primers, and PromoterFinder™ libraries to walk genomic DNA(Clontech, Palo Alto, Calif.). This procedure avoids the need to screenlibraries and is useful in finding intron/exon junctions. For allPCR-based methods, primers may be designed using commercially availablesoftware, such as OLIGO™ 4.06 primer analysis software (NationalBiosciences Inc., Plymouth, Minn.) or another appropriate program, to beabout 22 to 30 nucleotides in length, to have a GC content of about 50%or more, and to anneal to the template at temperatures of about 68° C.to 72° C.

When screening for full-length cDNAs, it is preferable to use librariesthat have been size-selected to include larger cDNAs. In addition,random-primed libraries, which often include sequences containing the 5'regions of genes, are preferable for situations in which an oligo d(T)library does not yield a full-length cDNA. Genomic libraries may beuseful for extension of sequence into 5' non-transcribed regulatoryregions.

Capillary electrophoresis systems which are commercially available maybe used to analyze the size or confirm the nucleotide sequence ofsequencing or PCR products. In particular, capillary sequencing mayemploy flowable polymers for electrophoretic separation, four differentnucleotide-specific, laser-stimulated fluorescent dyes, and a chargecoupled device camera for detection of the emitted wavelengths.Output/light intensity may be converted to electrical signal usingappropriate software (e.g., Genotyper™ and Sequence Navigator™, PerkinElmer), and the entire process from loading of samples to computeranalysis and electronic data display may be computer controlled.Capillary electrophoresis is especially preferable for sequencing smallDNA fragments which may be present in limited amounts in a particularsample.

In another embodiment of the invention, polynucleotide sequences orfragments thereof which encode HUTRAN may be cloned in recombinant DNAmolecules that direct expression of HUTRAN, or fragments or functionalequivalents thereof, in appropriate host cells. Due to the inherentdegeneracy of the genetic code, other DNA sequences which encodesubstantially the same or a functionally equivalent amino acid sequencemay be produced and used to express HUTRAN.

The nucleotide sequences of the present invention can be engineeredusing methods generally known in the art in order to alterHUTRAN-encoding sequences for a variety of purposes including, but notlimited to, modification of the cloning, processing, and/or expressionof the gene product. DNA shuffling by random fragmentation and PCRreassembly of gene fragments and synthetic oligonucleotides may be usedto engineer the nucleotide sequences. For example,oligonucleotide-mediated site-directed mutagenesis may be used tointroduce mutations that create new restriction sites, alterglycosylation patterns, change codon preference, produce splicevariants, and so forth.

In another embodiment, sequences encoding HUTRAN may be synthesized, inwhole or in part, using chemical methods well known in the art. (See,e.g., Caruthers, M. H. et al. (1980) Nucl. Acids Res. Symp. Ser.215-223, and Horn, T. et al. (1980) Nucl. Acids Res. Symp. Ser.225-232.) Alternatively, HUTRAN itself or a fragment thereof may besynthesized using chemical methods. For example, peptide synthesis canbe performed using various solid-phase techniques. (See, e.g., Roberge,J. Y. et al. (1995) Science 269:202-204.) Automated synthesis may beachieved using the ABI 431A peptide synthesizer (Perkin Elmer).Additionally, the amino acid sequence of HUTRAN, or any part thereof,may be altered during direct synthesis and/or combined with sequencesfrom other proteins, or any part thereof, to produce a variantpolypeptide.

The peptide may be substantially purified by preparative highperformance liquid chromatography. (See, e.g, Chiez, R. M. and F. Z.Regnier (1990) Methods Enzymol. 182:392-421.) The composition of thesynthetic peptides may be confirmed by amino acid analysis or bysequencing. (See, e.g., Creighton, T. (1984) Proteins, Structures andMolecular Properties, WH Freeman and Co., New York, N.Y.)

In order to express a biologically active HUTRAN, the nucleotidesequences encoding HUTRAN or derivatives thereof may be inserted into anappropriate expression vector, i.e., a vector which contains thenecessary elements for transcriptional and translational control of theinserted coding sequence in a suitable host. These elements includeregulatory sequences, such as enhancers, constitutive and induciblepromoters, and 5' and 3' untranslated regions in the vector and inpolynucleotide sequences encoding HUTRAN. Such elements may vary intheir strength and specificity. Specific initiation signals may also beused to achieve more efficient translation of sequences encoding HUTRAN.Such signals include the ATG initiation codon and adjacent sequences,e.g. the Kozak sequence. In cases where sequences encoding HUTRAN andits initiation codon and upstream regulatory sequences are inserted intothe appropriate expression vector, no additional transcriptional ortranslational control signals may be needed. However, in cases whereonly coding sequence, or a fragment thereof, is inserted, exogenoustranslational control signals including an in-frame ATG initiation codonshould be provided by the vector. Exogenous translational elements andinitiation codons may be of various origins, both natural and synthetic.The efficiency of expression may be enhanced by the inclusion ofenhancers appropriate for the particular host cell system used. (See,e.g., Scharf, D. et al. (1994) Results Probl. Cell Differ. 20:125-162.)

Methods which are well known to those skilled in the art may be used toconstruct expression vectors containing sequences encoding HUTRAN andappropriate transcriptional and translational control elements. Thesemethods include in vitro recombinant DNA techniques, synthetictechniques, and in vivo genetic recombination. (See, e.g., Sambrook, J.et al. (1989) Molecular Cloning, A Laboratory Manual, Cold Spring HarborPress, Plainview, N.Y., ch. 4, 8, and 16-17; and Ausubel, F. M. et al.(1995, and periodic supplements) Current Protocols in Molecular Biology,John Wiley & Sons, New York, N.Y., ch. 9, 13, and 16.)

A variety of expression vector/host systems may be utilized to containand express sequences encoding HUTRAN. These include, but are notlimited to, microorganisms such as bacteria transformed with recombinantbacteriophage, plasmid, or cosmid DNA expression vectors; yeasttransformed with yeast expression vectors; insect cell systems infectedwith viral expression vectors (e.g., baculovirus); plant cell systemstransformed with viral expression vectors (e.g., cauliflower mosaicvirus (CaMV) or tobacco mosaic virus (TMV)) or with bacterial expressionvectors (e.g., Ti or pBR322 plasmids); or animal cell systems. Theinvention is not limited by the host cell employed.

In bacterial systems, a number of cloning and expression vectors may beselected depending upon the use intended for polynucleotide sequencesencoding HUTRAN. For example, routine cloning, subcloning, andpropagation of polynucleotide sequences encoding HUTRAN can be achievedusing a multifunctional E. coli vector such as Bluescript® (Stratagene)or pSport1™ plasmid (Life Technologies, Inc., Gaithersburg, Md.).Ligation of sequences encoding HUTRAN into the vector's multiple cloningsite disrupts the lacZ gene, allowing a colorimetric screening procedurefor identification of transformed bacteria containing recombinantmolecules. In addition, these vectors may be useful for in vitrotranscription, dideoxy sequencing, single strand rescue with helperphage, and creation of nested deletions in the cloned sequence. (See,e.g., Van Heeke, G. and S. M. Schuster (1989) J. Biol. Chem.264:5503-5509.) When large quantities of HUTRAN are needed, e.g. for theproduction of antibodies, vectors which direct high level expression ofHUTRAN may be used. For example, vectors containing the strong,inducible T5 or T7 bacteriophage promoter may be used.

Yeast expression systems may be used for production of HUTRAN. A numberof vectors containing constitutive or inducible promoters, such as alphafactor, alcohol oxidase, and PGH, may be used in the yeast Saccharomycescerevisiae or Pichia pastoris. In addition, such vectors direct eitherthe secretion or intracellular retention of expressed proteins andenable integration of foreign sequences into the host genome for stablepropagation. (See, e.g., Ausubel, supra; and Grant et al. (1987) MethodsEnzymol. 153:516-54; Scorer, C. A. et al. (1994) Bio/Technology12:181-184.)

Plant systems may also be used for expression of HUTRAN. Transcriptionof sequences encoding HUTRAN may be driven viral promoters, e.g., the35S and 19S promoters of CaMV used alone or in combination with theomega leader sequence from TMV. (Takamatsu, N. (1987) EMBO J.6:307-311.) Alternatively, plant promoters such as the small subunit ofRUBISCO or heat shock promoters may be used. (See, e.g., Coruzzi, G. etal. (1984) EMBO J. 3:1671-1680; Broglie, R. et al. (1984) Science224:838-843; and Winter, J. et al. (1991) Results Probl. Cell Differ.17:85-105.) These constructs can be introduced into plant cells bydirect DNA transformation or pathogen-mediated transfection. (See, e.g.,Hobbs, S. or Murry, L. E. in McGraw Hill Yearbook of Science andTechnology (1992) McGraw Hill, New York, N.Y.; pp. 191-196.)

In mammalian cells, a number of viral-based expression systems may beutilized. In cases where an adenovirus is used as an expression vector,sequences encoding HUTRAN may be ligated into an adenovirustranscription/translation complex consisting of the late promoter andtripartite leader sequence. Insertion in a non-essential E1 or E3 regionof the viral genome may be used to obtain infective virus whichexpresses HUTRAN in host cells. (See, e.g., Logan, J. and T. Shenk(1984) Proc. Natl. Acad. Sci. 81:3655-3659.) In addition, transcriptionenhancers, such as the Rous sarcoma virus (RSV) enhancer, may be used toincrease expression in mammalian host cells. SV40 or EBV-based vectorsmay also be used for high-level protein expression.

Human artificial chromosomes (HACs) may also be employed to deliverlarger fragments of DNA than can be contained in and expressed from aplasmid. HACs of about 6 kb to 10 Mb are constructed and delivered viaconventional delivery methods (liposomes, polycationic amino polymers,or vesicles) for therapeutic purposes.

For long term production of recombinant proteins in mammalian systems,stable expression of HUTRAN in cell lines is preferred. For example,sequences encoding HUTRAN can be transformed into cell lines usingexpression vectors which may contain viral origins of replication and/orendogenous expression elements and a selectable marker gene on the sameor on a separate vector. Following the introduction of the vector, cellsmay be allowed to grow for about 1 to 2 days in enriched media beforebeing switched to selective media. The purpose of the selectable markeris to confer resistance to a selective agent, and its presence allowsgrowth and recovery of cells which successfully express the introducedsequences. Resistant clones of stably transformed cells may bepropagated using tissue culture techniques appropriate to the cell type.

Any number of selection systems may be used to recover transformed celllines. These include, but are not limited to, the herpes simplex virusthymidine kinase and adenine phosphoribosyltransferase genes, for use intk⁻ or apr⁻ cells, respectively. (See, e.g., Wigler, M. et al. (1977)Cell 11:223-232; and Lowy, I. et al. (1980) Cell 22:817-823.) Also,antimetabolite, antibiotic, or herbicide resistance can be used as thebasis for selection. For example, dhfr confers resistance tomethotrexate; neo confers resistance to the aminoglycosides neomycin andG-418; and als or pat confer resistance to chlorsulfuron andphosphinotricin acetyltransferase, respectively. (See, e.g., Wigler, M.et al. (1980) Proc. Natl. Acad. Sci. 77:3567-3570; Colbere-Garapin, F.et al (1981) J. Mol. Biol. 150:1-14; and Murry, supra.) Additionalselectable genes have been described, e.g., trpB and hisD, which altercellular requirements for metabolites. (See, e.g., Hartman, S. C. and R.C. Mulligan (1988) Proc. Natl. Acad. Sci. 85:8047-8051.) Visiblemarkers, e.g., anthocyanins, green fluorescent proteins (GFP) (Clontech,Palo Alto, Calif.), β glucuronidase and its substrate β-D-glucuronoside,or luciferase and its substrate luciferin may be used. These markers canbe used not only to identify transformants, but also to quantify theamount of transient or stable protein expression attributable to aspecific vector system. (See, e.g., Rhodes, C. A. et al. (1995) MethodsMol. Biol. 55:121-131.)

Although the presence/absence of marker gene expression suggests thatthe gene of interest is also present, the presence and expression of thegene may need to be confirmed. For example, if the sequence encodingHUTRAN is inserted within a marker gene sequence, transformed cellscontaining sequences encoding HUTRAN can be identified by the absence ofmarker gene function. Alternatively, a marker gene can be placed intandem with a sequence encoding HUTRAN under the control of a singlepromoter. Expression of the marker gene in response to induction orselection usually indicates expression of the tandem gene as well.

In general, host cells that contain the nucleic acid sequence encodingHUTRAN and that express HUTRAN may be identified by a variety ofprocedures known to those of skill in the art. These procedures include,but are not limited to, DNA-DNA or DNA-RNA hybridizations, PCRamplification, and protein bioassay or immunoassay techniques whichinclude membrane, solution, or chip based technologies for the detectionand/or quantification of nucleic acid or protein sequences.

Immunological methods for detecting and measuring the expression ofHUTRAN using either specific polyclonal or monoclonal antibodies areknown in the art. Examples of such techniques include enzyme-linkedimmunosorbent assays (ELISAs), radioimmunoassays (RIAs), andfluorescence activated cell sorting (FACS). A two-site, monoclonal-basedimmunoassay utilizing monoclonal antibodies reactive to twonon-interfering epitopes on HUTRAN is preferred, but a competitivebinding assay may be employed. These and other assays are well known inthe art. (See, e.g., Hampton, R. et al. (1990) Serological Methods, aLaboratory Manual, APS Press, St Paul, Minn., Section IV; Coligan, J. E.et al. (1997 and periodic supplements) Current Protocols in Immunology,Greene Pub. Associates and Wiley-Interscience, New York, N.Y.; andMaddox, D. E. et al. (1983) J. Exp. Med. 158:1211-1216).

A wide variety of labels and conjugation techniques are known by thoseskilled in the art and may be used in various nucleic acid and aminoacid assays. Means for producing labeled hybridization or PCR probes fordetecting sequences related to polynucleotides encoding HUTRAN includeoligolabeling, nick translation, end-labeling, or PCR amplificationusing a labeled nucleotide. Alternatively, the sequences encodingHUTRAN, or any fragments thereof, may be cloned into a vector for theproduction of an mRNA probe. Such vectors are known in the art, arecommercially available, and may be used to synthesize RNA probes invitro by addition of an appropriate RNA polymerase such as T7, T3, orSP6 and labeled nucleotides. These procedures may be conducted using avariety of commercially available kits, such as those provided byAmersham Pharmacia Biotech (Piscataway, N.J.), Promega (Madison, Wis.),and U.S. Biochemical Corp. (Cleveland, Ohio). Suitable reportermolecules or labels which may be used for ease of detection includeradionuclides, enzymes, fluorescent, chemiluminescent, or chromogenicagents, as well as substrates, cofactors, inhibitors, magneticparticles, and the like.

Host cells transformed with nucleotide sequences encoding HUTRAN may becultured under conditions suitable for the expression and recovery ofthe protein from cell culture. The protein produced by a transformedcell may be secreted or retained intracellularly depending on thesequence and/or the vector used. As will be understood by those of skillin the art, expression vectors containing polynucleotides which encodeHUTRAN may be designed to contain signal sequences which directsecretion of HUTRAN through a prokaryotic or eukaryotic cell membrane.

In addition, a host cell strain may be chosen for its ability tomodulate expression of the inserted sequences or to process theexpressed protein in the desired fashion. Such modifications of thepolypeptide include, but are not limited to, acetylation, carboxylation,glycosylation, phosphorylation, lipidation, and acylation.Post-translational processing which cleaves a "prepro" form of theprotein may also be used to specify protein targeting, folding, and/oractivity. Different host cells which have specific cellular machineryand characteristic mechanisms for post-translational activities (e.g.,CHO, HeLa, MDCK, HEK293, and W138), are available from the American TypeCulture Collection (ATCC, Manassas, VA.) and may be chosen to ensure thecorrect modification and processing of the foreign protein.

In another embodiment of the invention, natural, modified, orrecombinant nucleic acid sequences encoding HUTRAN may be ligated to aheterologous sequence resulting in translation of a fusion protein inany of the aforementioned host systems. For example, a chimeric HUTRANprotein containing a heterologous moiety that can be recognized by acommercially available antibody may facilitate the screening of peptidelibraries for inhibitors of HUTRAN activity. Heterologous protein andpeptide moieties may also facilitate purification of fusion proteinsusing commercially available affinity matrices. Such moieties include,but are not limited to, glutathione S-transferase (GST), maltose bindingprotein (MBP), thioredoxin (Trx), calmodulin binding peptide (CBP),6-His, FLAG, c-myc, and hemagglutinin (HA). GST, MBP, Trx, CBP, and6-His enable purification of their cognate fusion proteins onimmobilized glutathione, maltose, phenylarsine oxide, calmodulin, andmetal-chelate resins, respectively. FLAG, c-myc, and hemagglutinin (HA)enable immunoaffinity purification of fusion proteins using commerciallyavailable monoclonal and polyclonal antibodies that specificallyrecognize these epitope tags. A fusion protein may also be engineered tocontain a proteolytic cleavage site located between the HUTRAN encodingsequence and the heterologous protein sequence, so that HUTRAN may becleaved away from the heterologous moiety following purification.Methods for fusion protein expression and purification are discussed inAusubel, F. M. et al. (1995 and periodic supplements) Current Protocolsin Molecular Biology, John Wiley & Sons, New York, N.Y., ch 10. Avariety of commercially available kits may also be used to facilitateexpression and purification of fusion proteins.

In a further embodiment of the invention, synthesis of radiolabeledHUTRAN may be achieved in vitro using the TNT™ rabbit reticulocytelysate or wheat germ extract systems (Promega, Madison, Wis.). Thesesystems couple transcription and translation of protein-coding sequencesoperably associated with the T7, T3, or SP6 promoters. Translation takesplace in the presence of a radiolabeled amino acid precursor, preferably³⁵ S-methionine.

Fragments of HUTRAN may be produced not only by recombinant production,but also by direct peptide synthesis using solid-phase techniques. (See,e.g., Creighton, supra pp. 55-60.) Protein synthesis may be performed bymanual techniques or by automation. Automated synthesis may be achieved,for example, using the Applied Biosystems 431A peptide synthesizer(Perkin Elmer). Various fragments of HUTRAN may be synthesizedseparately and then combined to produce the full length molecule.

Therapeutics

Chemical and structural similarity, e.g., in the context of sequencesand motifs, exists between HUTRAN-1 and glutamine-phenylpyruvateaminotransferase from man (GI 758591), between HUTRAN-2 andkynurenine/α-aminoadipate aminotransferase from rat (GI 1050752), andbetween HUTRAN-3 and arginine methyltransferase from man (GI 1808648).In addition, HUTRAN is expressed in cancerous, inflamed, male and femalereproductive, nervous, and gastrointestinal tissues. Therefore, HUTRANappears to play a role in autoimmune/inflammatory, neurological,reproductive, and gastrointestinal disorders, and cancer.

Therefore, in one embodiment, an antagonist of HUTRAN may beadministered to a subject to treat or prevent an autoimmune/inflammatorydisorder. Such an autoimmune/inflammatory disorder may include, but isnot limited to, acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout,Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritablebowel syndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma. In oneaspect, an antibody which specifically binds HUTRAN may be used directlyas an antagonist or indirectly as a targeting or delivery mechanism forbringing a pharmaceutical agent to cells or tissue which express HUTRAN.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding HUTRAN may be administered to a subject to treator prevent an autoimmune/inflammatory disorder including, but notlimited to, those described above.

In another embodiment, HUTRAN or a fragment or derivative thereof may beadministered to a subject to treat or prevent a neurological disorder.Such neurological disorders can include, but are not limited to,epilepsy, ischemic cerebrovascular disease, stroke, cerebral neoplasms,Alzheimer's disease, Pick's disease, Huntington's disease, dementia,Parkinson's disease and other extrapyramidal disorders, amyotrophiclateral sclerosis and other motor neuron disorders, progressive neuralmuscular atrophy, retinitis pigmentosa, hereditary ataxias, multiplesclerosis and other demyelinating diseases, bacterial and viralmeningitis, brain abscess, subdural empyema, epidural abscess,suppurative intracranial thrombophlebitis, myelitis and radiculitis,viral central nervous system disease; prion diseases including kuru,Creutzfeldt-Jakob disease, and Gerstmann-Straussler-Scheinker syndrome;fatal familial insomnia, nutritional and metabolic diseases of thenervous system, neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis; inherited, metabolic,endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis;mental disorders including mood, anxiety, and schizophrenic disorders;akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette'sdisorder.

In another embodiment, a vector capable of expressing HUTRAN or afragment or derivative thereof may be administered to a subject to treator prevent a neurological disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUTRAN in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a neurological disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHUTRAN may be administered to a subject to treat or prevent aneurological disorder including, but not limited to, those listed above.

In another embodiment, HUTRAN or a fragment or derivative thereof may beadministered to a subject to treat or prevent a reproductive disorder.Such reproductive disorders can include, but are not limited to,disorders of prolactin production; infertility, including tubal disease,ovulatory defects, and endometriosis; disruptions of the estrous cycle,disruptions of the menstrual cycle, polycystic ovary syndrome, ovarianhyperstimulation syndrome, endometrial and ovarian tumors, uterinefibroids, autoimmune disorders, ectopic pregnancies, and teratogenesis;cancer of the breast, fibrocystic breast disease, and galactorrhea;disruptions of spermatogenesis, abnormal sperm physiology, cancer of thetestis, cancer of the prostate, benign prostatic hyperplasia,prostatitis, Peyronie's disease, carcinoma of the male breast, andgynecomastia.

In another embodiment, a vector capable of expressing HUTRAN or afragment or derivative thereof may be administered to a subject to treator prevent a reproductive disorder including, but not limited to, thosedescribed above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUTRAN in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a reproductive disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHUTRAN may be administered to a subject to treat or prevent areproductive disorder including, but not limited to, those listed above.

In another embodiment, HUTRAN or a fragment or derivative thereof may beadministered to a subject to treat or prevent a gastrointestinaldisorder. Such gastrointestinal disorders can include, but are notlimited to, dysphagia, peptic esophagitis, esophageal spasm, esophagealstricture, esophageal carcinoma, dyspepsia, indigestion, gastritis,gastric carcinoma, anorexia, nausea, emesis, gastroparesis, antral orpyloric edema, abdominal angina, pyrosis, gastroenteritis, intestinalobstruction, infections of the intestinal tract, peptic ulcer,cholelithiasis, cholecystitis, cholestasis, pancreatitis, pancreaticcarcinoma, biliary tract disease, hepatitis, hyperbilirubinemia,cirrhosis, passive congestion of the liver, hepatoma, infectiouscolitis, ulcerative colitis, ulcerative proctitis, Crohn's disease,Whipple's disease, Mallory-Weiss syndrome, colonic carcinoma, colonicobstruction, irritable bowel syndrome, short bowel syndrome, diarrhea,constipation, gastrointestinal hemorrhage, and acquired immunodeficiencysyndrome (AIDS) enteropathy.

In another embodiment, a vector capable of expressing HUTRAN or afragment or derivative thereof may be administered to a subject to treator prevent a gastrointestinal disorder including, but not limited to,those described above.

In a further embodiment, a pharmaceutical composition comprising asubstantially purified HUTRAN in conjunction with a suitablepharmaceutical carrier may be administered to a subject to treat orprevent a gastrointestinal disorder including, but not limited to, thoseprovided above.

In still another embodiment, an agonist which modulates the activity ofHUTRAN may be administered to a subject to treat or prevent agastrointestinal disorder including, but not limited to, those listedabove.

In another embodiment, an antagonist of HUTRAN may be administered to asubject to treat or prevent a cancer. Such a cancer may include, but isnot limited to, adenocarcinoma, leukemia, lymphoma, melanoma, myeloma,sarcoma, teratocarcinoma, and, in particular, cancers of the adrenalgland, bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. In one aspect, an antibodywhich specifically binds HUTRAN may be used directly as an antagonist orindirectly as a targeting or delivery mechanism for bringing apharmaceutical agent to cells or tissue which express HUTRAN.

In an additional embodiment, a vector expressing the complement of thepolynucleotide encoding HUTRAN may be administered to a subject to treator prevent a cancer including, but not limited to, those describedabove.

In other embodiments, any of the proteins, antagonists, antibodies,agonists, complementary sequences, or vectors of the invention may beadministered in combination with other appropriate therapeutic agents.Selection of the appropriate agents for use in combination therapy maybe made by one of ordinary skill in the art, according to conventionalpharmaceutical principles. The combination of therapeutic agents may actsynergistically to effect the treatment or prevention of the variousdisorders described above. Using this approach, one may be able toachieve therapeutic efficacy with lower dosages of each agent, thusreducing the potential for adverse side effects.

An antagonist of HUTRAN may be produced using methods which aregenerally known in the art. In particular, purified HUTRAN may be usedto produce antibodies or to screen libraries of pharmaceutical agents toidentify those which specifically bind HUTRAN. Antibodies to HUTRAN mayalso be generated using methods that are well known in the art. Suchantibodies may include, but are not limited to, polyclonal, monoclonal,chimeric, and single chain antibodies, Fab fragments, and fragmentsproduced by a Fab expression library. Neutralizing antibodies (i.e.,those which inhibit dimer formation) are especially preferred fortherapeutic use.

For the production of polyclonal antibodies, various hosts includinggoats, rabbits, rats, mice, humans, and others may be immunized byinjection with HUTRAN or with any fragment or oligopeptide thereof whichhas immunogenic properties. Rats and mice are preferred hosts fordownstream applications involving monoclonal antibody production.Depending on the host species, various adjuvants may be used to increaseimmunological response. Such adjuvants include, but are not limited to,Freund's, mineral gels such as aluminum hydroxide, and surface activesubstances such as lysolecithin, pluronic polyols, polyanions, peptides,oil emulsions, KLH, and dinitrophenol. Among adjuvants used in humans,BCG (bacilli Calmette-Guerin) and Corynebacterium parvum are especiallypreferable. (For review of methods for antibody production and analysis,see, e.g., Harlow, E. and Lane, D. (1988) Antibodies: A LaboratoryManual, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.).

It is preferred that the oligopeptides, peptides, or fragments used toinduce antibodies to HUTRAN have an amino acid sequence consisting of atleast about 5 amino acids, and, more preferably, of at least about 14amino acids. It is also preferable that these oligopeptides, peptides,or fragments are identical to a portion of the amino acid sequence ofthe natural protein and contain the entire amino acid sequence of asmall, naturally occurring molecule. Short stretches of HUTRAN aminoacids may be fused with those of another protein, such as KLH, andantibodies to the chimeric molecule may be produced.

Monoclonal antibodies to HUTRAN may be prepared using any techniquewhich provides for the production of antibody molecules by continuouscell lines in culture. These include, but are not limited to, thehybridoma technique, the human B-cell hybridoma technique, and theEBV-hybridoma technique. (See, e.g., Kohler, G. et al. (1975) Nature256:495-497; Kozbor, D. et al. (1985) J. Immunol. Methods 81:31-42;Cote, R. J. et al. (1983) Proc. Natl. Acad. Sci. 80:2026-2030; and Cole,S. P. et al. (1984) Mol. Cell Biol. 62:109-120.)

In addition, techniques developed for the production of "chimericantibodies," such as the splicing of mouse antibody genes to humanantibody genes to obtain a molecule with appropriate antigen specificityand biological activity, can be used. (See, e.g., Morrison, S. L. et al.(1984) Proc. Natl. Acad. Sci. 81:6851-6855; Neuberger, M. S. et al.(1984) Nature 312:604-608; and Takeda, S. et al. (1985) Nature314:452-454.) Alternatively, techniques described for the production ofsingle chain antibodies may be adapted, using methods known in the art,to produce HUTRAN-specific single chain antibodies. Antibodies withrelated specificity, but of distinct idiotypic composition, may begenerated by chain shuffling from random combinatorial immunoglobulinlibraries. (See, e.g., Burton D. R. (1991) Proc. Natl. Acad. Sci.88:10134-10137.)

Antibodies may also be produced by inducing in vivo production in thelymphocyte population or by screening immunoglobulin libraries or panelsof highly specific binding reagents as disclosed in the literature.(See, e.g., Orlandi, R. et al. (1989) Proc. Natl. Acad. Sci. 86:3833-3837; and Winter, G. et al. (1991) Nature 349:293-299.)

Antibody fragments which contain specific binding sites for HUTRAN mayalso be generated. For example, such fragments include, but are notlimited to, F(ab')2 fragments produced by pepsin digestion of theantibody molecule and Fab fragments generated by reducing the disulfidebridges of the F(ab')2 fragments. Alternatively, Fab expressionlibraries may be constructed to allow rapid and easy identification ofmonoclonal Fab fragments with the desired specificity. (See, e.g., Huse,W. D. et al. (1989) Science 246:1275-1281.)

Various immunoassays may be used for screening to identify antibodieshaving the desired specificity and minimal cross-reactivity. Numerousprotocols for competitive binding or immunoradiometric assays usingeither polyclonal or monoclonal antibodies with establishedspecificities are well known in the art. Such immunoassays typicallyinvolve the measurement of complex formation between HUTRAN and itsspecific antibody. A two-site, monoclonal-based immunoassay utilizingmonoclonal antibodies reactive to two non-interfering HUTRAN epitopes ispreferred, but a competitive binding assay may also be employed.(Maddox, supra.)

Various methods such as Scatchard analysis in conjunction withradioimmunoassay techniques may be used to assess the affinity ofantibodies for HUTRAN. Affinity is expressed as an association constant,K_(a), which is defined as the molar concentration of HUTRAN-antibodycomplex divided by the molar concentrations of free antigen and freeantibody under equilibrium conditions. The K_(a) determined for apreparation of polyclonal antibodies, which are heterogeneous in theiraffinities for multiple HUTRAN epitopes, represents the averageaffinity, or avidity, of the antibodies for HUTRAN. The K_(a) determinedfor a preparation of monoclonal antibodies, which are monospecific for aparticular HUTRAN epitope, represents a true measure of affinity.High-affinity antibody preparations with K_(a) ranging from about 10⁹ to10¹² L/mole are preferred for use in immunoassays in which theHUTRAN-antibody complex must withstand rigorous manipulations.Low-affinity antibody preparations with K_(a) ranging from about 10⁶ to10⁷ L/mole are preferred for use in immunopurification and similarprocedures which ultimately require dissociation of HUTRAN, preferablyin active form, from the antibody. (Catty, D. (1988) Antibodies. VolumeI: A Practical Approach, IRL Press, Washington, D. C.; and Liddell, J.E. and Cryer, A. (1991) A Practical Guide to Monoclonal Antibodies, JohnWiley & Sons, New York, N.Y.).

The titre and avidity of polyclonal antibody preparations may be furtherevaluated to determine the quality and suitability of such preparationsfor certain downstream applications. For example, a polyclonal antibodypreparation containing at least 1-2 mg specific antibody/ml, preferably5-10 mg specific antibody/ml, is preferred for use in proceduresrequiring precipitation of HUTRAN-antibody complexes. Procedures forevaluating antibody specificity, titer, and avidity, and guidelines forantibody quality and usage in various applications, are generallyavailable. (See, e.g., Catty, supra, and Coligan et al. supra.)

In another embodiment of the invention, the polynucleotides encodingHUTRAN, or any fragment or complement thereof, may be used fortherapeutic purposes. In one aspect, the complement of thepolynucleotide encoding HUTRAN may be used in situations in which itwould be desirable to block the transcription of the mRNA. Inparticular, cells may be transformed with sequences complementary topolynucleotides encoding HUTRAN. Thus, complementary molecules orfragments may be used to modulate HUTRAN activity, or to achieveregulation of gene function. Such technology is now well known in theart, and sense or antisense oligonucleotides or larger fragments can bedesigned from various locations along the coding or control regions ofsequences encoding HUTRAN.

Expression vectors derived from retroviruses, adenoviruses, or herpes orvaccinia viruses, or from various bacterial plasmids, may be used fordelivery of nucleotide sequences to the targeted organ, tissue, or cellpopulation. Methods which are well known to those skilled in the art canbe used to construct vectors to express nucleic acid sequencescomplementary to the polynucleotides encoding HUTRAN. (See, e.g.,Sambrook, supra; and Ausubel, supra.)

Genes encoding HUTRAN can be turned off by transforming a cell or tissuewith expression vectors which express high levels of a polynucleotide,or fragment thereof, encoding HUTRAN. Such constructs may be used tointroduce untranslatable sense or antisense sequences into a cell. Evenin the absence of integration into the DNA, such vectors may continue totranscribe RNA molecules until they are disabled by endogenousnucleases. Transient expression may last for a month or more with anon-replicating vector, and may last even longer if appropriatereplication elements are part of the vector system.

As mentioned above, modifications of gene expression can be obtained bydesigning complementary sequences or antisense molecules (DNA, RNA, orPNA) to the control, 5', or regulatory regions of the gene encodingHUTRAN. Oligonucleotides derived from the transcription initiation site,e.g., between about positions -10 and +10 from the start site, arepreferred. Similarly, inhibition can be achieved using triple helixbase-pairing methodology. Triple helix pairing is useful because itcauses inhibition of the ability of the double helix to opensufficiently for the binding of polymerases, transcription factors, orregulatory molecules. Recent therapeutic advances using triplex DNA havebeen described in the literature. (See, e.g., Gee, J. E. et al. (1994)in Huber, B. E. and B. I. Carr, Molecular and Immunologic Approaches,Futura Publishing Co., Mt. Kisco, N.Y., pp. 163-177.) A complementarysequence or antisense molecule may also be designed to block translationof mRNA by preventing the transcript from binding to ribosomes.

Ribozymes, enzymatic RNA molecules, may also be used to catalyze thespecific cleavage of RNA. The mechanism of ribozyme action involvessequence-specific hybridization of the ribozyme molecule tocomplementary target RNA, followed by endonucleolytic cleavage. Forexample, engineered hammerhead motif ribozyme molecules may specificallyand efficiently catalyze endonucleolytic cleavage of sequences encodingHUTRAN.

Specific ribozyme cleavage sites within any potential RNA target areinitially identified by scanning the target molecule for ribozymecleavage sites, including the following sequences: GUA, GUU, and GUC.Once identified, short RNA sequences of between 15 and 20ribonucleotides, corresponding to the region of the target genecontaining the cleavage site, may be evaluated for secondary structuralfeatures which may render the oligonucleotide inoperable. Thesuitability of candidate targets may also be evaluated by testingaccessibility to hybridization with complementary oligonucleotides usingribonuclease protection assays.

Complementary ribonucleic acid molecules and ribozymes of the inventionmay be prepared by any method known in the art for the synthesis ofnucleic acid molecules. These include techniques for chemicallysynthesizing oligonucleotides such as solid phase phosphoramiditechemical synthesis. Alternatively, RNA molecules may be generated by invitro and in vivo transcription of DNA sequences encoding HUTRAN. SuchDNA sequences may be incorporated into a wide variety of vectors withsuitable RNA polymerase promoters such as T7 or SP6. Alternatively,these cDNA constructs that synthesize complementary RNA, constitutivelyor inducibly, can be introduced into cell lines, cells, or tissues.

RNA molecules may be modified to increase intracellular stability andhalf-life. Possible modifications include, but are not limited to, theaddition of flanking sequences at the 5' and/or 3' ends of the molecule,or the use of phosphorothioate or 2' O-methyl rather thanphosphodiesterase linkages within the backbone of the molecule. Thisconcept is inherent in the production of PNAs and can be extended in allof these molecules by the inclusion of nontraditional bases such asinosine, queosine, and wybutosine, as well as acetyl-, methyl-, thio-,and similarly modified forms of adenine, cytidine, guanine, thymine, anduridine which are not as easily recognized by endogenous endonucleases.

Many methods for introducing vectors into cells or tissues are availableand equally suitable for use in vivo, in vitro, and ex vivo. For ex vivotherapy, vectors may be introduced into stem cells taken from thepatient and clonally propagated for autologous transplant back into thatsame patient. Delivery by transfection, by liposome injections, or bypolycationic amino polymers may be achieved using methods which are wellknown in the art. (See, e.g., Goldman, C. K. et al. (1997) NatureBiotechnology 15:462-466.)

Any of the therapeutic methods described above may be applied to anysubject in need of such therapy, including, for example, mammals such asdogs, cats, cows, horses, rabbits, monkeys, and most preferably, humans.

An additional embodiment of the invention relates to the administrationof a pharmaceutical or sterile composition, in conjunction with apharmaceutically acceptable carrier, for any of the therapeutic effectsdiscussed above. Such pharmaceutical compositions may consist of HUTRAN,antibodies to HUTRAN, and mimetics, agonists, antagonists, or inhibitorsof HUTRAN. The compositions may be administered alone or in combinationwith at least one other agent, such as a stabilizing compound, which maybe administered in any sterile, biocompatible pharmaceutical carrierincluding, but not limited to, saline, buffered saline, dextrose, andwater. The compositions may be administered to a patient alone, or incombination with other agents, drugs, or hormones.

The pharmaceutical compositions utilized in this invention may beadministered by any number of routes including, but not limited to,oral, intravenous, intramuscular, intra-arterial, intramedullary,intrathecal, intraventricular, transdermal, subcutaneous,intraperitoneal, intranasal, enteral, topical, sublingual, or rectalmeans.

In addition to the active ingredients, these pharmaceutical compositionsmay contain suitable pharmaceutically-acceptable carriers comprisingexcipients and auxiliaries which facilitate processing of the activecompounds into preparations which can be used pharmaceutically. Furtherdetails on techniques for formulation and administration may be found inthe latest edition of Remington's Pharmaceutical Sciences (MaackPublishing Co., Easton, Pa.).

Pharmaceutical compositions for oral administration can be formulatedusing pharmaceutically acceptable carriers well known in the art indosages suitable for oral administration. Such carriers enable thepharmaceutical compositions to be formulated as tablets, pills, dragees,capsules, liquids, gels, syrups, slurries, suspensions, and the like,for ingestion by the patient.

Pharmaceutical preparations for oral use can be obtained throughcombining active compounds with solid excipient and processing theresultant mixture of granules (optionally, after grinding) to obtaintablets or dragee cores. Suitable auxiliaries can be added, if desired.Suitable excipients include carbohydrate or protein fillers, such assugars, including lactose, sucrose, mannitol, and sorbitol; starch fromcorn, wheat, rice, potato, or other plants; cellulose, such as methylcellulose, hydroxypropylmethyl-cellulose, or sodiumcarboxymethylcellulose; gums, including arabic and tragacanth; andproteins, such as gelatin and collagen. If desired, disintegrating orsolubilizing agents may be added, such as the cross-linked polyvinylpyrrolidone, agar, and alginic acid or a salt thereof, such as sodiumalginate.

Dragee cores may be used in conjunction with suitable coatings, such asconcentrated sugar solutions, which may also contain gum arabic, talc,polyvinylpyrrolidone, carbopol gel, polyethylene glycol, and/or titaniumdioxide, lacquer solutions, and suitable organic solvents or solventmixtures. Dyestuffs or pigments may be added to the tablets or drageecoatings for product identification or to characterize the quantity ofactive compound, i.e., dosage.

Pharmaceutical preparations which can be used orally include push-fitcapsules made of gelatin, as well as soft, sealed capsules made ofgelatin and a coating, such as glycerol or sorbitol. Push-fit capsulescan contain active ingredients mixed with fillers or binders, such aslactose or starches, lubricants, such as talc or magnesium stearate,and, optionally, stabilizers. In soft capsules, the active compounds maybe dissolved or suspended in suitable liquids, such as fatty oils,liquid, or liquid polyethylene glycol with or without stabilizers.

Pharmaceutical formulations suitable for parenteral administration maybe formulated in aqueous solutions, preferably in physiologicallycompatible buffers such as Hanks' solution, Ringer's solution, orphysiologically buffered saline. Aqueous injection suspensions maycontain substances which increase the viscosity of the suspension, suchas sodium carboxymethyl cellulose, sorbitol, or dextran. Additionally,suspensions of the active compounds may be prepared as appropriate oilyinjection suspensions. Suitable lipophilic solvents or vehicles includefatty oils, such as sesame oil, or synthetic fatty acid esters, such asethyl oleate, triglycerides, or liposomes. Non-lipid polycationic aminopolymers may also be used for delivery. Optionally, the suspension mayalso contain suitable stabilizers or agents to increase the solubilityof the compounds and allow for the preparation of highly concentratedsolutions.

For topical or nasal administration, penetrants appropriate to theparticular barrier to be permeated are used in the formulation. Suchpenetrants are generally known in the art.

The pharmaceutical compositions of the present invention may bemanufactured in a manner that is known in the art, e.g., by means ofconventional mixing, dissolving, granulating, dragee-making, levigating,emulsifying, encapsulating, entrapping, or lyophilizing processes.

The pharmaceutical composition may be provided as a salt and can beformed with many acids, including but not limited to, hydrochloric,sulfuric, acetic, lactic, tartaric, malic, and succinic acid. Salts tendto be more soluble in aqueous or other protonic solvents than are thecorresponding free base forms. In other cases, the preferred preparationmay be a lyophilized powder which may contain any or all of thefollowing: 1 mM to 50 mM histidine, 0.1% to 2% sucrose, and 2% to 7%mannitol, at a pH range of 4.5 to 5.5, that is combined with bufferprior to use.

After pharmaceutical compositions have been prepared, they can be placedin an appropriate container and labeled for treatment of an indicatedcondition. For administration of HUTRAN, such labeling would includeamount, frequency, and method of administration.

Pharmaceutical compositions suitable for use in the invention includecompositions wherein the active ingredients are contained in aneffective amount to achieve the intended purpose. The determination ofan effective dose is well within the capability of those skilled in theart.

For any compound, the therapeutically effective dose can be estimatedinitially either in cell culture assays, e.g., of neoplastic cells or inanimal models such as mice, rats, rabbits, dogs, or pigs. An animalmodel may also be used to determine the appropriate concentration rangeand route of administration. Such information can then be used todetermine useful doses and routes for administration in humans.

A therapeutically effective dose refers to that amount of activeingredient, for example HUTRAN or fragments thereof, antibodies ofHUTRAN, and agonists, antagonists or inhibitors of HUTRAN, whichameliorates the symptoms or condition. Therapeutic efficacy and toxicitymay be determined by standard pharmaceutical procedures in cell culturesor with experimental animals, such as by calculating the ED₅₀ (the dosetherapeutically effective in 50% of the population) or LD₅₀ (the doselethal to 50% of the population) statistics. The dose ratio oftherapeutic to toxic effects is the therapeutic index, and it can beexpressed as the ED₅₀ /LD₅₀ ratio. Pharmaceutical compositions whichexhibit large therapeutic indices are preferred. The data obtained fromcell culture assays and animal studies are used to formulate a range ofdosage for human use. The dosage contained in such compositions ispreferably within a range of circulating concentrations that includesthe ED₅₀ with little or no toxicity. The dosage varies within this rangedepending upon the dosage form employed, the sensitivity of the patient,and the route of administration.

The exact dosage will be determined by the practitioner, in light offactors related to the subject requiring treatment. Dosage andadministration are adjusted to provide sufficient levels of the activemoiety or to maintain the desired effect. Factors which may be takeninto account include the severity of the disease state, the generalhealth of the subject, the age, weight, and gender of the subject, timeand frequency of administration, drug combination(s), reactionsensitivities, and response to therapy. Long-acting pharmaceuticalcompositions may be administered every 3 to 4 days, every week, orbiweekly depending on the half-life and clearance rate of the particularformulation.

Normal dosage amounts may vary from about 0.1 μg to 100,000 μg, up to atotal dose of about 1 gram, depending upon the route of administration.Guidance as to particular dosages and methods of delivery is provided inthe literature and generally available to practitioners in the art.Those skilled in the art will employ different formulations fornucleotides than for proteins or their inhibitors. Similarly, deliveryof polynucleotides or polypeptides will be specific to particular cells,conditions, locations, etc.

Diagnostics

In another embodiment, antibodies which specifically bind HUTRAN may beused for the diagnosis of disorders characterized by expression ofHUTRAN, or in assays to monitor patients being treated with HUTRAN oragonists, antagonists, or inhibitors of HUTRAN. Antibodies useful fordiagnostic purposes may be prepared in the same manner as describedabove for therapeutics. Diagnostic assays for HUTRAN include methodswhich utilize the antibody and a label to detect HUTRAN in human bodyfluids or in extracts of cells or tissues. The antibodies may be usedwith or without modification, and may be labeled by covalent ornon-covalent attachment of a reporter molecule. A wide variety ofreporter molecules, several of which are described above, are known inthe art and may be used.

A variety of protocols for measuring HUTRAN, including ELISAs, RIAs, andFACS, are known in the art and provide a basis for diagnosing altered orabnormal levels of HUTRAN expression. Normal or standard values forHUTRAN expression are established by combining body fluids or cellextracts taken from normal mammalian subjects, preferably human, withantibody to HUTRAN under conditions suitable for complex formation. Theamount of standard complex formation may be quantitated by variousmethods, preferably by photometric means. Quantities of HUTRAN expressedin subject samples from biopsied tissues are compared with the standardvalues. Deviation between standard and subject values establishes theparameters for diagnosing disease.

In another embodiment of the invention, the polynucleotides encodingHUTRAN may be used for diagnostic purposes. The polynucleotides whichmay be used include oligonucleotide sequences, complementary RNA and DNAmolecules, and PNAs. The polynucleotides may be used to detect andquantitate gene expression in biopsied tissues in which expression ofHUTRAN may be correlated with disease. The diagnostic assay may be usedto determine absence, presence, and excess expression of HUTRAN, and tomonitor regulation of HUTRAN levels during therapeutic intervention.

In one aspect, hybridization with PCR probes which are capable ofdetecting polynucleotide sequences, including genomic sequences,encoding HUTRAN or closely related molecules may be used to identifynucleic acid sequences which encode HUTRAN. The specificity of theprobe, whether it is made from a highly specific region, e.g., the 5'regulatory region, or from a less specific region, e.g., a conservedmotif, and the stringency of the hybridization or amplification(maximal, high, intermediate, or low), will determine whether the probeidentifies only naturally occurring sequences encoding HUTRAN, allelicvariants, or related sequences.

Probes may also be used for the detection of related sequences, andshould preferably have at least 50% sequence identity to any of theHUTRAN encoding sequences. The hybridization probes of the subjectinvention may be DNA or RNA and may be derived from the sequence of thesequences of SEQ ID NO:4, SEQ ID NO:5, or SEQ ID NO:6 or from genomicsequences including promoters, enhancers, and introns of the HUTRANgene.

Means for producing specific hybridization probes for DNAs encodingHUTRAN include the cloning of polynucleotide sequences encoding HUTRANor HUTRAN derivatives into vectors for the production of mRNA probes.Such vectors are known in the art, are commercially available, and maybe used to synthesize RNA probes in vitro by means of the addition ofthe appropriate RNA polymerases and the appropriate labeled nucleotides.Hybridization probes may be labeled by a variety of reporter groups, forexample, by radionuclides such as ³² P or ³⁵ S, or by enzymatic labels,such as alkaline phosphatase coupled to the probe via avidin/biotincoupling systems, and the like.

Polynucleotide sequences encoding HUTRAN may be used for the diagnosisof a disorder associated with expression of HUTRAN. Examples of such adisorder include, but are not limited to, autoimmune/inflammatorydisorders such as acquired immunodeficiency syndrome (AIDS), Addison'sdisease, adult respiratory distress syndrome, allergies, ankylosingspondylitis, amyloidosis, anemia, asthma, atherosclerosis, autoimmunehemolytic anemia, autoimmune thyroiditis, bronchitis, cholecystitis,contact dermatitis, Crohn's disease, atopic dermatitis, dermatomyositis,diabetes mellitus, emphysema, episodic lymphopenia withlymphocytotoxins, erythroblastosis fetalis, erythema nodosum, atrophicgastritis, glomerulonephritis, Goodpasture's syndrome, gout,Graves'disease, Hashimoto's thyroiditis, hypereosinophilia, irritablebowel syndrome, multiple sclerosis, myasthenia gravis, myocardial orpericardial inflammation, osteoarthritis, osteoporosis, pancreatitis,polymyositis, psoriasis, Reiter's syndrome, rheumatoid arthritis,scleroderma, Sjogren's syndrome, systemic anaphylaxis, systemic lupuserythematosus, systemic sclerosis, thrombocytopenic purpura, ulcerativecolitis, uveitis, Werner syndrome, complications of cancer,hemodialysis, and extracorporeal circulation, viral, bacterial, fungal,parasitic, protozoal, and helminthic infections, and trauma;neurological disorders such as epilepsy, ischemic cerebrovasculardisease, stroke, cerebral neoplasms, Alzheimer's disease, Pick'sdisease, Huntington's disease, dementia, Parkinson's disease and otherextrapyramidal disorders, amyotrophic lateral sclerosis and other motorneuron disorders, progressive neural muscular atrophy, retinitispigmentosa, hereditary ataxias, multiple sclerosis and otherdemyelinating diseases, bacterial and viral meningitis, brain abscess,subdural empyema, epidural abscess, suppurative intracranialthrombophlebitis, myelitis and radiculitis, viral central nervous systemdisease; prion diseases including kuru, Creutzfeldt-Jakob disease, andGerstmann-Straussler-Scheinker syndrome; fatal familial insomnia,nutritional and metabolic diseases of the nervous system,neurofibromatosis, tuberous sclerosis, cerebelloretinalhemangioblastomatosis, encephalotrigeminal syndrome, mental retardationand other developmental disorders of the central nervous system,cerebral palsy, neuroskeletal disorders, autonomic nervous systemdisorders, cranial nerve disorders, spinal cord diseases, musculardystrophy and other neuromuscular disorders, peripheral nervous systemdisorders, dermatomyositis and polymyositis; inherited, metabolic,endocrine, and toxic myopathies; myasthenia gravis, periodic paralysis;mental disorders including mood, anxiety, and schizophrenic disorders;akathesia, amnesia, catatonia, diabetic neuropathy, tardive dyskinesia,dystonias, paranoid psychoses, postherpetic neuralgia, and Tourette'sdisorder; reproductive disorders such as disorders of prolactinproduction; infertility, including tubal disease, ovulatory defects, andendometriosis; disruptions of the estrous cycle, disruptions of themenstrual cycle, polycystic ovary syndrome, ovarian hyperstimulationsyndrome, endometrial and ovarian tumors, uterine fibroids, autoimmunedisorders, ectopic pregnancies, and teratogenesis; cancer of the breast,fibrocystic breast disease, and galactorrhea; disruptions ofspermatogenesis, abnormal sperm physiology, cancer of the testis, cancerof the prostate, benign prostatic hyperplasia, prostatitis, Peyronie'sdisease, carcinoma of the male breast, and gynecomastia;gastrointestinal disorders such as; dysphagia, peptic esophagitis,esophageal spasm, esophageal stricture, esophageal carcinoma, dyspepsia,indigestion, gastritis, gastric carcinoma, anorexia, nausea, emesis,gastroparesis, antral or pyloric edema, abdominal angina, pyrosis,gastroenteritis, intestinal obstruction, infections of the intestinaltract, peptic ulcer, cholelithiasis, cholecystitis, cholestasis,pancreatitis, pancreatic carcinoma, biliary tract disease, hepatitis,hyperbilirubinemia, cirrhosis, passive congestion of the liver,hepatoma, infectious colitis, ulcerative colitis, ulcerative proctitis,Crohn's disease, Whipple's disease, Mallory-Weiss syndrome, coloniccarcinoma, colonic obstruction, irritable bowel syndrome, short bowelsyndrome, diarrhea, constipation, gastrointestinal hemorrhage, andacquired immunodeficiency syndrome (AIDS) enteropathy; and cancers suchas adenocarcinoma, leukemia, lymphoma, melanoma, myeloma, sarcoma,teratocarcinoma, and, in particular, cancers of the adrenal gland,bladder, bone, bone marrow, brain, breast, cervix, gall bladder,ganglia, gastrointestinal tract, heart, kidney, liver, lung, muscle,ovary, pancreas, parathyroid, penis, prostate, salivary glands, skin,spleen, testis, thymus, thyroid, and uterus. The polynucleotidesequences encoding HUTRAN may be used in Southern or Northern analysis,dot blot, or other membrane-based technologies; in PCR technologies; indipstick, pin, and ELISA assays; and in microarrays utilizing fluids ortissues from patients to detect altered HUTRAN expression. Suchqualitative or quantitative methods are well known in the art.

In a particular aspect, the nucleotide sequences encoding HUTRAN may beuseful in assays that detect the presence of associated disorders,particularly those mentioned above. The nucleotide sequences encodingHUTRAN may be labeled by standard methods and added to a fluid or tissuesample from a patient under conditions suitable for the formation ofhybridization complexes. After a suitable incubation period, the sampleis washed and the signal is quantitated and compared with a standardvalue. If the amount of signal in the patient sample is significantlyaltered in comparison to a control sample then the presence of alteredlevels of nucleotide sequences encoding HUTRAN in the sample indicatesthe presence of the associated disorder. Such assays may also be used toevaluate the efficacy of a particular therapeutic treatment regimen inanimal studies, in clinical trials, or to monitor the treatment of anindividual patient.

In order to provide a basis for the diagnosis of a disorder associatedwith expression of HUTRAN, a normal or standard profile for expressionis established. This may be accomplished by combining body fluids orcell extracts taken from normal subjects, either animal or human, with asequence, or a fragment thereof, encoding HUTRAN, under conditionssuitable for hybridization or amplification. Standard hybridization maybe quantified by comparing the values obtained from normal subjects withvalues from an experiment in which a known amount of a substantiallypurified polynucleotide is used. Standard values obtained in this mannermay be compared with values obtained from samples from patients who aresymptomatic for a disorder. Deviation from standard values is used toestablish the presence of a disorder.

Once the presence of a disorder is established and a treatment protocolis initiated, hybridization assays may be repeated on a regular basis todetermine if the level of expression in the patient begins toapproximate that which is observed in the normal subject. The resultsobtained from successive assays may be used to show the efficacy oftreatment over a period ranging from several days to months.

With respect to cancer, the presence of a relatively high amount oftranscript in biopsied tissue from an individual may indicate apredisposition for the development of the disease, or may provide ameans for detecting the disease prior to the appearance of actualclinical symptoms. A more definitive diagnosis of this type may allowhealth professionals to employ preventative measures or aggressivetreatment earlier thereby preventing the development or furtherprogression of the cancer.

Additional diagnostic uses for oligonucleotides designed from thesequences encoding HUTRAN may involve the use of PCR. These oligomersmay be chemically synthesized, generated enzymatically, or produced invitro. Oligomers will preferably contain a fragment of a polynucleotideencoding HUTRAN, or a fragment of a polynucleotide complementary to thepolynucleotide encoding HUTRAN, and will be employed under optimizedconditions for identification of a specific gene or condition. Oligomersmay also be employed under less stringent conditions for detection orquantitation of closely related DNA or RNA sequences.

Methods which may also be used to quantitate the expression of HUTRANinclude radiolabeling or biotinylating nucleotides, coamplification of acontrol nucleic acid, and interpolating results from standard curves.(See, e.g., Melby, P. C. et al. (1993) J. Immunol. Methods 159:235-244;and Duplaa, C. et al. (1993) Anal. Biochem. 229-236.) The speed ofquantitation of multiple samples may be accelerated by running the assayin an ELISA format where the oligomer of interest is presented invarious dilutions and a spectrophotometric or colorimetric responsegives rapid quantitation.

In further embodiments, oligonucleotides or longer fragments derivedfrom any of the polynucleotide sequences described herein may be used astargets in a microarray. The microarray can be used to monitor theexpression level of large numbers of genes simultaneously and toidentify genetic variants, mutations, and polymorphisms. Thisinformation may be used to determine gene function, to understand thegenetic basis of a disorder, to diagnose a disorder, and to develop andmonitor the activities of therapeutic agents.

Microarrays may be prepared, used, and analyzed using methods known inthe art. (See, e.g., Brennan, T. M. et al. (1995) U.S. Pat. No.5,474,796; Schena, M. et al. (1996) Proc. Natl. Acad. Sci.93:10614-10619; Baldeschweiler et al. (1995) PCT applicationWO95/251116; Shalon, D. et al. (1995) PCT application WO95/35505;Heller, R. A. et al. (1997) Proc. Natl. Acad. Sci. 94:2150-2155; andHeller, M. J. et al. (1997) U.S. Pat. No. 5,605,662.)

In another embodiment of the invention, nucleic acid sequences encodingHUTRAN may be used to generate hybridization probes useful in mappingthe naturally occurring genomic sequence. The sequences may be mapped toa particular chromosome, to a specific region of a chromosome, or toartificial chromosome constructions, e.g., human artificial chromosomes(HACs), yeast artificial chromosomes (YACs), bacterial artificialchromosomes (BACs), bacterial P1 constructions, or single chromosomecDNA libraries. (See, e.g., Price, C. M. (1993) Blood Rev. 7:127-134;and Trask, B. J. (1991) Trends Genet. 7:149-154.)

Fluorescent in situ hybridization (FISH) may be correlated with otherphysical chromosome mapping techniques and genetic map data. (See, e.g.,Heinz-Ulrich, et al. (1995) in Meyers, R. A. (ed.) Molecular Biology andBiotechnology, VCH Publishers New York, N.Y., pp. 965-968.) Examples ofgenetic map data can be found in various scientific journals or at theOnline Mendelian Inheritance in Man (OMIM) site. Correlation between thelocation of the gene encoding HUTRAN on a physical chromosomal map and aspecific disorder, or a predisposition to a specific disorder, may helpdefine the region of DNA associated with that disorder. The nucleotidesequences of the invention may be used to detect differences in genesequences among normal, carrier, and affected individuals.

In situ hybridization of chromosomal preparations and physical mappingtechniques, such as linkage analysis using established chromosomalmarkers, may be used for extending genetic maps. Often the placement ofa gene on the chromosome of another mammalian species, such as mouse,may reveal associated markers even if the number or arm of a particularhuman chromosome is not known. New sequences can be assigned tochromosomal arms by physical mapping. This provides valuable informationto investigators searching for disease genes using positional cloning orother gene discovery techniques. Once the disease or syndrome has beencrudely localized by genetic linkage to a particular genomic region,e.g., ataxia-telangiectasia to 11q22-23, any sequences mapping to thatarea may represent associated or regulatory genes for furtherinvestigation. (See, e.g., Gatti, R. A. et al. (1988) Nature336:577-580.) The nucleotide sequence of the subject invention may alsobe used to detect differences in the chromosomal location due totranslocation, inversion, etc., among normal, carrier, or affectedindividuals.

In another embodiment of the invention, HUTRAN, its catalytic orimmunogenic fragments, or oligopeptides thereof can be used forscreening libraries of compounds in any of a variety of drug screeningtechniques. The fragment employed in such screening may be free insolution, affixed to a solid support, borne on a cell surface, orlocated intracellularly. The formation of binding complexes betweenHUTRAN and the agent being tested may be measured.

Another technique for drug screening provides for high throughputscreening of compounds having suitable binding affinity to the proteinof interest. (See, e.g., Geysen, et al. (1984) PCT applicationWO084/03564.) In this method, large numbers of different small testcompounds are synthesized on a solid substrate, such as plastic pins orsome other surface. The test compounds are reacted with HUTRAN, orfragments thereof, and washed. Bound HUTRAN is then detected by methodswell known in the art. Purified HUTRAN can also be coated directly ontoplates for use in the aforementioned drug screening techniques.Alternatively, non-neutralizing antibodies can be used to capture thepeptide and immobilize it on a solid support.

In another embodiment, one may use competitive drug screening assays inwhich neutralizing antibodies capable of binding HUTRAN specificallycompete with a test compound for binding HUTRAN. In this manner,antibodies can be used to detect the presence of any peptide whichshares one or more antigenic determinants with HUTRAN.

In additional embodiments, the nucleotide sequences which encode HUTRANmay be used in any molecular biology techniques that have yet to bedeveloped, provided the new techniques rely on properties of nucleotidesequences that are currently known, including, but not limited to, suchproperties as the triplet genetic code and specific base pairinteractions.

The examples below are provided to illustrate the subject invention andare not included for the purpose of limiting the invention.

EXAMPLES

I. Construction of cDNA Libraries

RNA was isolated from tissues described in Table 4. Some tissues werehomogenized and lysed in guanidinium isothiocyanate, while others werehomogenized and lysed in phenol or in a suitable mixture of denaturants,such as TRIZOL™ (Life Technologies, Inc., Gaithersburg, Md.), amonophasic solution of phenol and guanidine isothiocyanate. Theresulting lysates were centrifuged over CsCl cushions or extracted withchloroform. RNA was precipitated from the lysates with eitherisopropanol or sodium acetate and ethanol, or by other routine methods.

Phenol extraction and precipitation of RNA were repeated as necessary toincrease RNA purity. In some cases, RNA was treated with DNase. For mostlibraries, poly(A+) RNA was isolated using oligo d(T)-coupledparamagnetic particles (Promega Corp., Madison, Wis.), OLIGOTEX™ latexparticles (QIAGEN Inc., Valencia, Calif.), or an OLIGOTEX™ mRNApurification kit (QIAGEN Inc., Valencia, Calif.). Alternatively, RNA wasisolated directly from tissue lysates using other RNA isolation kits,e.g., the POLY(A)PURE™ mRNA purification kit (Ambion, Austin, Tex.).

cDNA was synthesized and cDNA libraries were constructed with theSUPERSCRIPT™ plasmid system (Life Technologies, Inc., Gaithersburg,Md.), using the recommended procedures or similar methods known in theart. (See, e.g., Ausubel, supra, 1997, units 5.1-6.6) Reversetranscription was initiated using oligo d(T) or random primers.Synthetic oligonucleotide adapters were ligated to double stranded cDNA,and the cDNA was digested with the appropriate restriction enzyme orenzymes. For most libraries, the cDNA was size-selected (300-1000 bp)using SEPHACRYL® S1000, SEPHAROSE® CL2B, or SEPHAROSE® CL4B columnchromatography (Amersham Pharmacia Biotech, Piscataway, N.J.) orpreparative agarose gel electrophoresis. cDNAs were ligated intocompatible restriction enzyme sites of the polylinker of pINCY (IncytePharmaceuticals, Inc., Palo Alto, Calif.). Recombinant plasmids weretransformed into competent E. coli cells, e.g., the XL1-Blue,XL1-BlueMRF, or SOLR™ strains (Stratagene, Inc., La Jolla, Calif.), orDH5α™, DH10B, or ElectroMAX DH10B (Life Technologies, Inc.,Gaithersburg, Md.).

II. Isolation of cDNA Clones

Plasmids were recovered from host cells by cell lysis. Plasmids werepurified using at least one of the following: a Magic or WIZARD®Minipreps DNA purification system (Promega Corp., Madison, Wis.); anAGTC® Miniprep purification kit (Edge Biosystems, Gaithersburg, Md.);the QIAWELL® 8 Plasmid, QIAWELL® 8 Plus Plasmid, or the QIAWELL® 8 UltraPlasmid purification systems (QIAGEN Inc., Valencia, Calif.); or theR.E.A.L.™ Prep 96 plasmid kit (QIAGEN Inc., Valencia, Calif.). Followingprecipitation, plasmids were resuspended in 0.1 ml of distilled waterand stored, with or without lyophilization, at 4° C.

Alternatively, plasmid DNA was amplified from host cell lysates usingdirect link PCR in a high-throughput format. (Rao, V. B. (1994) Anal.Biochem. 216:1-14.) Host cell lysis and thermal cycling steps werecarried out in a single reaction mixture. Samples were processed andstored in 384-well plates, and the concentration of amplified plasmidDNA was quantified fluorometrically using PICOGREEN® dye (MolecularProbes, Inc., Eugene, Oreg.) and a Fluoroskan II fluorescence scanner(Labsystems Oy, Helsinki, Finland).

III. Sequencing and Analysis

The cDNAs were prepared for sequencing using either an ABI CATALYST 800(Perkin Elmer) or a MICROLAB 2200 (Hamilton, Reno, Nev.) in combinationwith Peltier thermal cyclers (PTC200; MJ Research, Watertown Mass.). ThecDNAs were sequenced on the ABI 373 or 377 DNA sequencing systems(Perkin Elmer) by the method of Sanger F. and A. R. Coulson (1975; J.Mol. Biol. 94:441-448) using standard ABI protocols, base callingsoftware, and kits. Alternatively, cDNAs were sequenced using solutionsand dyes from Amersham Pharmacia Biotech. Reading frame was determinedusing standard methods (Ausubel, supra).

The cDNA sequences presented in Table 1 and the full length nucleotideand amino acid sequences disclosed in the Sequence Listing were queriedagainst databases such as GenBank primate (pri), rodent (rod), mammalian(mamp), vertebrate (vrtp), and eukaryote (eukp) databases, SwissProt,BLOCKS, and other databases which contain previously identified andannotated motifs and sequences. Algorithms such as Smith Waterman whichdeal with primary sequence patterns and secondary structure gappenalties (Smith, T. et al. (1992) Protein Engineering 5:35-51) andprograms and algorithms such as BLAST (Basic Local Alignment SearchTool; Altschul, S. F. (1993) J. Mol. Evol 36:290-300; and Altschul etal. (1990) J. Mol. Biol. 215:403-410), and HMM (Hidden Markov Models;Eddy, S. R. (1996) Cur. Opin. Str. Biol. 6:361-365 and Sonnhammer, E. L.L. et al. (1997) Proteins 28:405-420) were used to assemble and analyzenucleotide and amino acid sequences. The databases, programs,algorithms, methods and tools are available, well known in the art, anddescribed in Ausubel (supra, unit 7.7), in Meyers, R. A. (1995;Molecular Biology and Biotechnology, Wiley VCH, Inc, New York N.Y., p856-853), in documentation provided with software (Genetics ComputerGroup (GCG), Madison Wis.), and on the world wide web (www). Twocomprehensive websites which list, describe, and/or link many of thedatabases and tools are: 1) the www resource in practical sequenceanalysis (http://genome.wustl.edu/), and 2) the bibliography ofcomputational gene recognition (http://linkage.rockefeller.edu/wli/gene/programs.html). For example, the first website links PFAM as a database(http://genome.wustl.edu/Pfam/) and as an HMM search tool(http://genome.wustl.edu/eddy/cgi-bin/hmm₁₃ page.cgi).

Table 5 summarizes the databases and tools used herein.

IV. Northern Analysis

Northern analysis is a laboratory technique used to detect the presenceof a transcript of a gene and involves the hybridization of a labelednucleotide sequence to a membrane on which RNAs from a particular celltype or tissue have been bound. (See, e.g., Sambrook, supra, ch. 7; andAusubel, supra, ch. 4 and 16.)

Analogous computer techniques applying BLAST are used to search foridentical or related molecules in nucleotide databases such as GenBankor LIFESEQ™ database (Incyte Pharmaceuticals). This analysis is muchfaster than multiple membrane-based hybridizations. In addition, thesensitivity of the computer search can be modified to determine whetherany particular match is categorized as exact or similar.

The basis of the search is the product score, which is defined as:##EQU1## The product score takes into account both the degree ofsimilarity between two sequences and the length of the sequence match.For example, with a product score of 40, the match will be exact withina 1% to 2% error, and, with a product score of 70, the match will beexact. Similar molecules are usually identified by selecting those whichshow product scores between 15 and 40, although lower scores mayidentify related molecules.

The results of Northern analysis are reported as a list of libraries inwhich the transcript encoding HUTRAN occurs. Abundance and percentabundance are also reported. Abundance directly reflects the number oftimes a particular transcript is represented in a cDNA library, andpercent abundance is abundance divided by the total number of sequencesexamined in the cDNA library.

V. Extension of HUTRAN Encoding Polynucleotides

The nucleic acid sequences of Incyte Clones 1815528, 2150892, and2525071 were used to design oligonucleotide primers for extendingpartial nucleotide sequences to full length. For each nucleic acidsequence, one primer was synthesized to initiate extension of anantisense polynucleotide, and the other was synthesized to initiateextension of a sense polynucleotide. Primers were used to facilitate theextension of the known sequence "outward" generating ampliconscontaining new unknown nucleotide sequence for the region of interest.The initial primers were designed from the cDNA using OLIGO™ 4.06software (National Biosciences, Plymouth, Minn.), or another appropriateprogram, to be about 22 to 30 nucleotides in length, to have a GCcontent of about 50% or more, and to anneal to the target sequence attemperatures of about 68° C. to about 72° C.. Any stretch of nucleotideswhich would result in hairpin structures and primer-primer dimerizationswas avoided.

Selected human cDNA libraries (Life Technologies, Inc., Gaithersburg,Md.) were used to extend the sequence. If more than one extension isnecessary or desired, additional sets of primers are designed to furtherextend the known region.

High fidelity amplification was obtained by following the instructionsfor the XL-PCR™ kit (Perkin Elmer) and thoroughly mixing the enzyme andreaction mix. PCR was performed using the Peltier Thermal Cycler(PTC200; M. J. Research, Watertown, Mass.), beginning with 40 pmol ofeach primer and the recommended concentrations of all other componentsof the kit, with the following parameters:

    ______________________________________                                        Step 1   94° C. for 1 min (initial denaturation)                                 Step 2 65° C. for 1 min                                        Step 3 68° C. for 6 min                                                Step 4 94° C. for 15 sec                                               Step 5 65° C. for 1 min                                                Step 6 68° C. for 7 min                                                Step 7 Repeat steps 4 through 6 for an additional 15 cycles                   Step 8 94° C. for 15 sec                                               Step 9 65° C. for 1 min                                                Step 10 68° C. for 7:15 min                                            Step 11 Repeat steps 8 through 10 for an additional 12 cycles                 Step 12 72° C. for 8 min                                               Step 13 4° C. (and holding)                                          ______________________________________                                    

A 5 μl to 10 μl aliquot of the reaction mixture was analyzed byelectrophoresis on a low concentration (about 0.6% to 0.8%) agarosemini-gel to determine which reactions were successful in extending thesequence. Bands thought to contain the largest products were excisedfrom the gel, purified using QIAQUICK™ (QIAGEN Inc.), and trimmed ofoverhangs using Klenow enzyme to facilitate religation and cloning.

After ethanol precipitation, the products were redissolved in 13 μl ofligation buffer, 1 μl T4-DNA ligase (15 units) and 1 μl T4polynucleotide kinase were added, and the mixture was incubated at roomtemperature for 2 to 3 hours, or overnight at 16° C. Competent E. colicells (in 40 μl of appropriate media) were transformed with 3 μl ofligation mixture and cultured in 80 μl of SOC medium. (See, e.g.,Sambrook, supra, Appendix A, p. 2.) After incubation for one hour at 37°C., the E. coli mixture was plated on Luria Bertani (LB) agar (See,e.g., Sambrook, supra, Appendix A, p. 1) containing carbenicillin(2×carb). The following day, several colonies were randomly picked fromeach plate and cultured in 150 μl of liquid LB/2× carb medium placed inan individual well of an appropriate commercially-available sterile96-well microtiter plate. The following day, 5 μl of each overnightculture was transferred into a non-sterile 96-well plate and, afterdilution 1:10 with water, 5 μl from each sample was transferred into aPCR array.

For PCR amplification, 18 μl of concentrated PCR reaction mix (3.3×)containing 4 units of rTth DNA polymerase, a vector primer, and one orboth of the gene specific primers used for the extension reaction wereadded to each well. Amplification was performed using the followingconditions:

    ______________________________________                                        Step 1   94° C. for 60 sec                                               Step 2 94° C. for 20 sec                                               Step 3 55° C. for 30 sec                                               Step 4 72° C. for 90 sec                                               Step 5 Repeat steps 2 through 4 for an additional 29 cycles                   Step 6 72° C. for 180 sec                                              Step 7 4° C. (and holding)                                           ______________________________________                                    

Aliquots of the PCR reactions were run on agarose gels together withmolecular weight markers.

The sizes of the PCR products were compared to the original partialcDNAs, and appropriate clones were selected, ligated into plasmid, andsequenced.

In like manner, the nucleotide sequences of SEQ ID NO:4-6 are used toobtain 5' regulatory sequences using the procedure above,oligonucleotides designed for 5' extension, and an appropriate genomiclibrary.

VI. Labeling and Use of Individual Hybridization Probes

Hybridization probes derived from SEQ ID NO:4-6 are employed to screencDNAs, genomic DNAs, or mRNAs. Although the labeling ofoligonucleotides, consisting of about 20 base pairs, is specificallydescribed, essentially the same procedure is used with larger nucleotidefragments. Oligonucleotides are designed using state-of-the-art softwaresuch as OLIGO™ 4.06 software (National Biosciences) and labeled bycombining 50 pmol of each oligomer, 250 μCi of [γ-³² P] adenosinetriphosphate (Amersham, Chicago, Ill.), and T4 polynucleotide kinase(DuPont NEN®, Boston, Mass.). The labeled oligonucleotides aresubstantially purified using a Sephadex™ G-25 superfine size exclusiondextran bead column (Pharmacia & Upjohn, Kalamazoo, Mich.). An aliquotcontaining 10⁷ counts per minute of the labeled probe is used in atypical membrane-based hybridization analysis of human genomic DNAdigested with one of the following endonucleases: Ase I, Bgl II, Eco RI,Pst I, Xbal, or Pvu II (DuPont NEN, Boston, Mass.).

The DNA from each digest is fractionated on a 0.7% agarose gel andtransferred to nylon membranes (Nytran Plus, Schleicher & Schuell,Durham, N.H.). Hybridization is carried out for 16 hours at 40° C. Toremove nonspecific signals, blots are sequentially washed at roomtemperature under increasingly stringent conditions up to 0.1×salinesodium citrate and 0.5% sodium dodecyl sulfate. After XOMAT AR™ film(Kodak, Rochester, N.Y.) is exposed to the blots, hybridization patternsare compared visually.

VII. Microarrays

A chemical coupling procedure and an ink jet device can be used tosynthesize array elements on the surface of a substrate. (See, e.g.,Baldeschweiler, supra.) An array analogous to a dot or slot blot mayalso be used to arrange and link elements to the surface of a substrateusing thermal, UV, chemical, or mechanical bonding procedures. A typicalarray may be produced by hand or using available methods and machinesand contain any appropriate number of elements. After hybridization,nonhybridized probes are removed and a scanner used to determine thelevels and patterns of fluorescence. The degree of complementarity andthe relative abundance of each probe which hybridizes to an element onthe microarray may be assessed through analysis of the scanned images.

Full-length cDNAs, Expressed Sequence Tags (ESTs), or fragments thereofmay comprise the elements of the microarray. Fragments suitable forhybridization can be selected using software well known in the art suchas LASERGENE™. Full-length cDNAs, ESTs, or fragments thereofcorresponding to one of the nucleotide sequences of the presentinvention, or selected at random from a cDNA library relevant to thepresent invention, are arranged on an appropriate substrate, e.g., aglass slide. The cDNA is fixed to the slide using, e.g., UVcross-linking followed by thermal and chemical treatments and subsequentdrying. (See, e.g., Schena, M. et al. (1995) Science 270:467-470; andShalon, D. et al. (1996) Genome Res. 6:639-645.) Fluorescent probes areprepared and used for hybridization to the elements on the substrate.The substrate is analyzed by procedures described above.

VIII. Complementary Polynucleotides

Sequences complementary to the HUTRAN-encoding sequences, or any partsthereof, are used to detect, decrease, or inhibit expression ofnaturally occurring HUTRAN. Although use of oligonucleotides comprisingfrom about 15 to 30 base pairs is described, essentially the sameprocedure is used with smaller or with larger sequence fragments.Appropriate oligonucleotides are designed using OLIGO™ 4.06 software andthe coding sequence of HUTRAN. To inhibit transcription, a complementaryoligonucleotide is designed from the most unique 5' sequence and used toprevent promoter binding to the coding sequence. To inhibit translation,a complementary oligonucleotide is designed to prevent ribosomal bindingto the HUTRAN-encoding transcript.

IX. Expression of HUTRAN

Expression and purification of HUTRAN is achieved using bacterial orvirus-based expression systems. For expression of HUTRAN in bacteria,cDNA is subcloned into an appropriate vector containing an antibioticresistance gene and an inducible promoter that directs high levels ofcDNA transcription. Examples of such promoters include, but are notlimited to, the trp-lac (tac) hybrid promoter and the T5 or T7bacteriophage promoter in conjunction with the lac operator regulatoryelement. Recombinant vectors are transformed into suitable bacterialhosts, e.g., BL21 (DE3). Antibiotic resistant bacteria express HUTRANupon induction with isopropyl beta-D-thiogalactopyranoside (IPTG).Expression of HUTRAN in eukaryotic cells is achieved by infecting insector mammalian cell lines with recombinant Autographica californicanuclear polyhedrosis virus (AcMNPV), commonly known as baculovirus. Thenonessential polyhedrin gene of baculovirus is replaced with cDNAencoding HUTRAN by either homologous recombination or bacterial-mediatedtransposition involving transfer plasmid intermediates. Viralinfectivity is maintained and the strong polyhedrin promoter drives highlevels of cDNA transcription. Recombinant baculovirus is used to infectSpodoptera frugiperda (Sf9) insect cells in most cases, or humanhepatocytes, in some cases. Infection of the latter requires additionalgenetic modifications to baculovirus. (See Engelhard, E. K. et al.(1994) Proc. Natl. Acad. Sci. U.S.A. 91:3224-3227; Sandig, V. et al.(1996) Hum. Gene Ther. 7:1937-1945.)

In most expression systems, HUTRAN is synthesized as a fusion proteinwith, e.g., glutathione S-transferase (GST) or a peptide epitope tag,such as FLAG or 6-His, permitting rapid, single-step, affinity-basedpurification of recombinant fusion protein from crude cell lysates. GST,a 26-kilodalton enzyme from Schistosoma japonicum, enables thepurification of fusion proteins on immobilized glutathione underconditions that maintain protein activity and antigenicity (Pharmacia,Piscataway, N.J.). Following purification, the GST moiety can beproteolytically cleaved from HUTRAN at specifically engineered sites.FLAG, an 8-amino acid peptide, enables immunoaffinity purification usingcommercially available monoclonal and polyclonal anti-FLAG antibodies(Eastman Kodak, Rochester, N.Y.). 6-His, a stretch of six consecutivehistidine residues, enables purification on metal-chelate resins (QIAGENInc, Chatsworth, Calif.). Methods for protein expression andpurification are discussed in Ausubel, F. M. et al. (1995 and periodicsupplements) Current Protocols in Molecular Biology, John Wiley & Sons,New York, N.Y., ch 10, 16. Purified HUTRAN obtained by these methods canbe used directly in the following activity assay.

X. Demonstration of HUTRAN Activity

HUTRAN-1

HUTRAN-1 activity may be demonstrated by the ability to convertL-phenylalanine and α-keto-γ-methiolbutyrate to phenylpyruvate andL-methionine. (Cooper, A. J. L. and Meister, A. (1985) Meth. Enzymol.113:344-349.) The amount of phenylpyruvate formed is measured. Thereaction mixture contains 200 mM ammediol-HCl buffer (pH 9.0), 10 mML-phenylalanine, 5 mM α-keto-γ-methiolbutyrate, and HUTRAN-1 in a finalvolume of 0.1 ml. After incubating at 37° C. for 10 minutes, 0.9 ml of3.33 M NaOh is added. The absorbance at 322 nm, measured using aspectrophotometer, is proportional to the phentlpyruvate formed, andthus to the HUTRAN-1 in the starting sample. The absorbance due tophenylpyruvate is stable for at least 15 minutes.

HUTRAN-2

HUTRAN-2 activity may be demonstrated by the ability to convertL-glutamate and α-ketoadipate to α-aminoadipate and α-ketoglutarate.(Nakatani, supra.) The amount of α-ketoglutarate formed is measured. Thestandard assay contains, in a volume of 0.3 ml, 50 μmoles potassiumphosphate buffer (pH 7.5), 20 μg pyridoxal phosphate, 0.5 μmolesα-ketoadipate, and HUTRAN-2. After a 5 minute incubation at 37° C., thereaction is started by addition of 0.2 ml of 0.1 M potassium L-glutamateand allowed to proceed for 10 minutes at 37° C. The reaction(Reaction 1) is terminated by adding 0.1 ml of 1 M HCl. Afterneutralization of the mixture with 0.1 ml of 1 M KOH, a 0.3 ml aliquotis taken for the determination of the presence of α-ketoglutarate.α-Ketoglutarate is estimated by the amount of NADH oxidized in thepresence of NH₄ ⁺ and glutamate dehydrogenase. The estimation ofα-ketoglutarate is performed in a system consisting of 300 μmolespotassium phosphate buffer (pH 7.5), 150 μmoles NH₄ Cl, 0.3 μmole NADH,and the neutralized reaction mixture in a total volume of 3.0 ml. Thedecrease in absorbance at 340 nm after the addition of glutamatedehydrogenase, measured using a spectrophotometer, is proportional tothe α-ketoglutarate formed in Reaction 1, and thus to the HUTRAN-2 inthe starting sample.

HUTRAN-3

HUTRAN-3 activity may be demonstrated by the ability to methylate hnRNPA1 protein in vitro. (Lin, supra). The reaction contains 490 ngbacterially expressed recombinant human hnRNP A1 , 0.93 μM [³H]S-adenosyl-L-methionine (2.2 μCi), 2.0 μg HUTRAN-3, and buffer (25 mMTris-HCl, 1 mM EDTA, and 1 mM EGTA at pH 7.5) in a final volume of 30μl. The reaction mixtures are incubated at 30° C. for 30 minutes andthen subjected to SDS-polyacrylamide gel electrophoresis. The gel isstained with Coomassie Blue, dried and subjected to fluorography. Theposition of hnRNP A1 is determined by Coomassie Blue staining. Theamount of [³ H]methylated hnRNP A1, as determined by densitometry orPhosphorImager analysis (Molecular Dynamics, Sunnyvale, Calif.), isproportional to the amount of HUTRAN-3 in the starting sample.

XI. Functional Assays

HUTRAN function is assessed by expressing the sequences encoding HUTRANat physiologically elevated levels in mammalian cell culture systems.cDNA is subcloned into a mammalian expression vector containing a strongpromoter that drives high levels of cDNA expression. Vectors of choiceinclude pCMV SPOT™ (Life Technologies, Gaithersburg, Md.) and pCR™ 3.1(Invitrogen, Carlsbad, Calif., both of which contain the cytomegaloviruspromoter. 5-10 μg of recombinant vector are transiently transfected intoa human cell line, preferably of endothelial or hematopoietic origin,using either liposome formulations or electroporation. 1-2 μg of anadditional plasmid containing sequences encoding a marker protein areco-transfected. Expression of a marker protein provides a means todistinguish transfected cells from nontransfected cells and is areliable predictor of cDNA expression from the recombinant vector.Marker proteins of choice include, e.g., Green Fluorescent Protein (GFP)(Clontech, Palo Alto, Calif.), CD64, or a CD64-GFP fusion protein. Flowcytometry (FCM), an automated, laser optics-based technique, is used toidentify transfected cells expressing GFP or CD64-GFP, and to evaluateproperties, for example, their apoptotic state. FCM detects andquantifies the uptake of fluorescent molecules that diagnose eventspreceding or coincident with cell death. These events include changes innuclear DNA content as measured by staining of DNA with propidiumiodide; changes in cell size and granularity as measured by forwardlight scatter and 90 degree side light scatter; down-regulation of DNAsynthesis as measured by decrease in bromodeoxyuridine uptake;alterations in expression of cell surface and intracellular proteins asmeasured by reactivity with specific antibodies; and alterations inplasma membrane composition as measured by the binding offluorescein-conjugated Annexin V protein to the cell surface. Methods inflow cytometry are discussed in Ormerod, M. G. (1994) Flow Cytometry,Oxford, New York, N.Y.

The influence of HUTRAN on gene expression can be assessed using highlypurified populations of cells transfected with sequences encoding HUTRANand either CD64 or CD64-GFP. CD64 and CD64-GFP are expressed on thesurface of transfected cells and bind to conserved regions of humanimmunoglobulin G (IgG). Transfected cells are efficiently separated fromnontransfected cells using magnetic beads coated with either human IgGor antibody against CD64 (DYNAL, Lake Success, N.Y.). mRNA can bepurified from the cells using methods well known by those of skill inthe art. Expression of mRNA encoding HUTRAN and other genes of interestcan be analyzed by Northern analysis or microarray techniques.

XII. Production of HUTRAN Specific Antibodies

HUTRAN substantially purified using polyacrylamide gel electrophoresis(PAGE)(see, e.g., Harrington, M. G. (1990) Methods Enzymol.182:488-495), or other purification techniques, is used to immunizerabbits and to produce antibodies using standard protocols.

Alternatively, the HUTRAN amino acid sequence is analyzed usingLASERGENE™ software (DNASTAR Inc.) to determine regions of highimmunogenicity, and a corresponding oligopeptide is synthesized and usedto raise antibodies by means known to those of skill in the art. Methodsfor selection of appropriate epitopes, such as those near the C-terminusor in hydrophilic regions are well described in the art. (See, e.g.,Ausubel supra, ch. 11.)

Typically, oligopeptides 15 residues in length are synthesized using anApplied Biosystems Peptide Synthesizer Model 431A using fmoc-chemistryand coupled to KLH (Sigma, St. Louis, Mo.) by reaction withN-maleimidobenzoyl-N-hydroxysuccinimide ester (MBS) to increaseimmunogenicity. (See, e.g., Ausubel supra.) Rabbits are immunized withthe oligopeptide-KLH complex in complete Freund's adjuvant. Resultingantisera are tested for antipeptide activity by, for example, bindingthe peptide to plastic, blocking with 1% BSA, reacting with rabbitantisera, washing, and reacting with radio-iodinated goat anti-rabbitIgG.

XIII. Purification of Naturally Occurring HUTRAN Using SpecificAntibodies

Naturally occurring or recombinant HUTRAN is substantially purified byimmunoaffinity chromatography using antibodies specific for HUTRAN. Animmunoaffinity column is constructed by covalently coupling anti-HUTRANantibody to an activated chromatographic resin, such as CNBr-activatedSepharose (Pharmacia & Upjohn). After the coupling, the resin is blockedand washed according to the manufacturer's instructions.

Media containing HUTRAN are passed over the immunoaffinity column, andthe column is washed under conditions that allow the preferentialabsorbance of HUTRAN (e.g., high ionic strength buffers in the presenceof detergent). The column is eluted under conditions that disruptantibody/HUTRAN binding (e.g., a buffer of pH 2 to pH 3, or a highconcentration of a chaotrope, such as urea or thiocyanate ion), andHUTRAN is collected.

XIV. Identification of Molecules Which Interact with HUTRAN

HUTRAN, or biologically active fragments thereof, are labeled with ¹²⁵ IBolton-Hunter reagent. (See, e.g., Bolton et al. (1973) Biochem. J.133:529.) Candidate molecules previously arrayed in the wells of amulti-well plate are incubated with the labeled HUTRAN, washed, and anywells with labeled HUTRAN complex are assayed. Data obtained usingdifferent concentrations of HUTRAN are used to calculate values for thenumber, affinity, and association of HUTRAN with the candidatemolecules.

Various modifications and variations of the described methods andsystems of the invention will be apparent to those skilled in the artwithout departing from the scope and spirit of the invention. Althoughthe invention has been described in connection with specific preferredembodiments, it should be understood that the invention as claimedshould not be unduly limited to such specific embodiments. Indeed,various modifications of the described modes for carrying out theinvention which are obvious to those skilled in molecular biology orrelated fields are intended to be within the scope of the followingclaims.

                                      TABLE 1                                     __________________________________________________________________________    Protein SEQ                                                                         Nucleotide SEQ                                                            ID NO: ID NO: Clone ID Library Fragments                                    __________________________________________________________________________    1     4       1815528                                                                            PROSNOT20                                                                            1815528H1 (PROSNOT20), 2880980F6 (UTRSTUT05),                                      1815528x12C1 (PROSNOT20), 1815528x17C1                                        (PROSNOT20), 1819092T6 (PROSNOT20),                                      269916F1                                                  (HNT2NOT01), 1717401F6 (UCMCNOT02)                                            (SEQ ID NO: 7-13)                                                         2 5 2150892 BRAINOT09 2150892H1 (BRAINOT09), SAGA00872F1, SAGA01877F1,                                     SAGA01269R1, SAGA02228F1, SAGA01614F1,                                        301251T6 (TESTNOT04)                                 (SEQ ID NO: 14-20)                                                        3 6 2525071 BRAITUT21 2525071H1 (BRAITUT21), 1889292H1 (BLADTUT07),                                        2525071F6 (BRAITUT21), SAEA10009P1,                                      SAEA03283F1,                                              SAEAO1931R1, 1253024T6 (LUNGFET03), 1664573F6                                 (BRSTNOT09), 1474156T1 (LUNGTUT03)                                            (SEQ ID NO: 21-29)                                                      __________________________________________________________________________

                                      TABLE 2                                     __________________________________________________________________________        Amino                                                                              Potential                                                                             Potential                                                      Seq ID Acid Phosphorylation Glycosylation   Analytical                        NO: Residues Sites Sites Signature Sequences Identification Methods         __________________________________________________________________________    1   454  S189 S408 S124 S277AGKTF (pyridoxal                                                                        glutamine-                                                                            BLAST                               S189 S378 S422  phosphate binding site) phenylpyruvate BLOCKS                                                              S437 S12 S23   aminotrans                                                  ferase PFAM                         S32 S36 Y338  M268 through V284  MOTIFS                                       Y395  (transmembrane sequence)  PFAM                                              MOTIFS                                                                        TM                                                                      2 425 T21 S170 S11  5260FSK (pyridoxal kynurenine/alpha- BLAST                  T190 S210  phosphate binding site) aminoadipate MOTIFS                           aminotransferase                                                         3 447 S286 S85 T127 N18 N69 N343 methyltransferase motifs: arginine                                                       BLAST                               S330 T355 T71 N384 V27LDVGCGSG (region I) methyltransferase MOTIFS                                                         S191  I49YAVE (post-regio                                                  n I)  SPSCAN                          E88QVDIIIS (region II)                                                        Y117LKPSGNMFP (region III)                                                    L165RGAA (post-region III)                                                    M1 through A46 (signal                                                        sequence)                                                               __________________________________________________________________________

                                      TABLE 3                                     __________________________________________________________________________    Seq ID NO:                                                                          Tissue Expression (Fraction of Total)                                                            Diseases or Conditions (Fraction of Total)                                                      Vector                             __________________________________________________________________________    1     Reproductive (0.302) Cardiovascular (0.163)                                                      Cancer (0.465) Inflammation (0.256) Fetal                                                       pINCY                                 Hematopoietic/Immune (0.116) (0.233) Trauma (0.093)                           Gastrointestinal (0.093) Nervous (0.070)                                     2 Reproductive (0.429) Nervous (0.214) Cancer (0.429) Fetal (0.214)                                                    Trauma pINCY                          Developmental (0.143) Gastrointestinal (0.214) Inflammation (0.1443)                                                    (0.143)                            3 Gastrointestinal (0.259) Reproductive Cancer (0.537) Fetal (0.185)                                                   Inflammation pINCY                    (0.241) Developmental (0.111) Nervous (0.167) Trauma (0.093)                  (0.111)                                                                    __________________________________________________________________________

                                      TABLE 4                                     __________________________________________________________________________    Protein SEQ                                                                     ID NO: Clone ID Library Library Comment                                     __________________________________________________________________________    1     1815528                                                                            PROSNOT20                                                                            PROSNOT20 Library was constructed using RNA isolated                          from diseased                                                    prostate tissue removed from a 65-year-old Caucasian male during a                         radical                                                          prostatectomy. Pathology indicated adenofibromatous hyperplasia.                               Pathology for the associated tumor tissue indicated                       an adenocarcinoma.                                            2 2150892 BRAINOT09 BRAINOT09 Library was constructed using RNA                               isolated from brain tissue                                       removed from a Caucasian male fetus who died at 23 weeks' gestation.       3 2525071 BRAITUT21 BRAITUT21 Library was constructed using RNA                               isolated from brain tumor                                        tissue removed from the midline frontal lobe of a 61-year-old                              Caucasian                                                        female during excision of a cerebral meningeal lesion. Pathology                           indicated                                                        subfrontal meningothelial meningioma with no atypia. One ethmoid and          cerebrovascular disease, senile demential, hyperlipidemia, benign                              hypertension, atherosclerotic coronary artery                             disease, congestive heart                                        failure, and breast cancer.                                              __________________________________________________________________________

                                      TABLE 5                                     __________________________________________________________________________    Program/algorithm                                                                      Databases                                                                          Description                   Useful Parameters                 __________________________________________________________________________    ESTs                                                                            Smith Waterman GenBank Local alignment algorithm for homology searching                                                 min length = 49 nt                     <12% uncalled bases                                                        FASTA GenBank Fast nucleotide sequence database searching program for                                                   UNIX, VMS                           BLAST GenBank Ultra-fast database searching program for UNIX, VMS C                                                     source Log likelihood for                                                         exact matches is                   ˜10.sup.-25 and for                                                     homologs >10.sup.-8                                                        Full Length                                                                   Phred  Reads trace data from sequencing runs, makes base calls for                                                      assembly                              of cDNA sequences, produces quality scores                                  Phrap  Quality-score based assembly program for shotgun sequences match                                                 >56                                    score >120                                                                 CONSED  Graphical tool for editing Phrap contigs                              GCG Assembly, GenBank Wisconsin Package Programs for the assembly,                                                      editing, and                        Motifs, Profilescan, PROSITE characterization of nucleotide sequences                                                    Spscan  Examines proteins                                                    for secretory, signal                                                         sequences >7 strong, 4.5-7                                                        suggestive                      GENEMARK  Statistical analysis of nucleotide sequences to identify open                                                 reading                               frame                                                                       BLAST GenBank Ultra-fast database searching program for UNIX, VMS C                                                     source score >1000, P < 1e-5                                                    SwissProt                         FASTX GenBank Fast amino acid sequence database searching program log                                                   likelihood >17                       SwissProt for UNIX, VMS                                                      BLIMPS BLOCKS Weighted matrix analysis for prediction of protein family                                                 >1300 strong, 1000-                  PRINTS  1300 suggestive, P < 1c-3                                            PFAM PROSITE Analyses sequences 3-60 amino acids long which correspond                                                  to Score >11 strong, 8-10                                                        highly conserved regions                                                   of a protein family suggestive      HMM  Probabilistic approaches and modeling of the primary structure                                                     Score >11 strong, 8-10                                                           of protein families                                                        suggestive                          McDNAsis Pro  Software for sequence analysis                                  LASERGENE  Software programs (EditSeq, MegAlign, PrimerSelect, Protean,         SeqMan, etc.) for sequence analysis                                       __________________________________________________________________________

    __________________________________________________________________________    #             SEQUENCE LISTING                                                   - -  - - (1) GENERAL INFORMATION:                                             - -    (iii) NUMBER OF SEQUENCES: 32                                          - -  - - (2) INFORMATION FOR SEQ ID NO: 1:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 454 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PROSNOT20                                                        (B) CLONE: 1815528                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #1:                           - - Met Phe Leu Ala Gln Arg Ser Leu Cys Ser Le - #u Ser Gly Arg Ala                           - #5                  - #10                  - #15           - - Lys Phe Leu Lys Thr Ile Ser Ser Ser Lys Il - #e Leu Gly Phe Ser                           20 - #                 25 - #                 30              - - Thr Ser Ala Lys Met Ser Leu Lys Phe Thr As - #n Ala Lys Arg Ile                           35 - #                 40 - #                 45              - - Glu Gly Leu Asp Ser Asn Val Trp Ile Glu Ph - #e Thr Lys Leu Ala                           50 - #                 55 - #                 60              - - Ala Asp Pro Ser Val Val Asn Leu Gly Gln Gl - #y Phe Pro Asp Ile                           65 - #                 70 - #                 75              - - Ser Pro Pro Thr Tyr Val Lys Glu Glu Leu Se - #r Lys Ile Ala Ala                           80 - #                 85 - #                 90              - - Ile Asp Ser Leu Asn Gln Tyr Thr Arg Gly Ph - #e Gly His Pro Ser                           95 - #                100 - #                105              - - Leu Val Lys Ala Leu Ser Tyr Leu Tyr Glu Ly - #s Leu Tyr Gln Lys                          110  - #               115  - #               120              - - Gln Ile Asp Ser Asn Lys Glu Ile Leu Val Th - #r Val Gly Ala Tyr                          125  - #               130  - #               135              - - Gly Ser Leu Phe Asn Thr Ile Gln Ala Leu Il - #e Asp Glu Gly Asp                          140  - #               145  - #               150              - - Glu Val Ile Leu Ile Val Pro Phe Tyr Asp Cy - #s Tyr Glu Pro Met                          155  - #               160  - #               165              - - Val Arg Met Ala Gly Ala Thr Pro Val Phe Il - #e Pro Leu Arg Ser                          170  - #               175  - #               180              - - Lys Pro Val Tyr Gly Lys Arg Trp Ser Ser Se - #r Asp Trp Thr Leu                          185  - #               190  - #               195              - - Asp Pro Gln Glu Leu Glu Ser Lys Phe Asn Se - #r Lys Thr Lys Ala                          200  - #               205  - #               210              - - Ile Ile Leu Asn Thr Pro His Asn Pro Leu Gl - #y Lys Val Tyr Asn                          215  - #               220  - #               225              - - Arg Glu Glu Leu Gln Val Ile Ala Asp Leu Cy - #s Ile Lys Tyr Asp                          230  - #               235  - #               240              - - Thr Leu Cys Ile Ser Asp Glu Val Tyr Glu Tr - #p Leu Val Tyr Ser                          245  - #               250  - #               255              - - Gly Asn Lys His Leu Lys Ile Ala Thr Phe Pr - #o Gly Met Trp Glu                          260  - #               265  - #               270              - - Arg Thr Ile Thr Ile Gly Ser Ala Gly Lys Th - #r Phe Ser Val Thr                          275  - #               280  - #               285              - - Gly Trp Lys Leu Gly Trp Ser Ile Gly Pro As - #n His Leu Ile Lys                          290  - #               295  - #               300              - - His Leu Gln Thr Val Gln Gln Asn Thr Ile Ty - #r Thr Cys Ala Thr                          305  - #               310  - #               315              - - Pro Leu Gln Glu Ala Leu Ala Gln Ala Phe Tr - #p Ile Asp Ile Lys                          320  - #               325  - #               330              - - Arg Met Asp Asp Pro Glu Cys Tyr Phe Asn Se - #r Leu Pro Lys Glu                          335  - #               340  - #               345              - - Leu Glu Val Lys Arg Asp Arg Met Val Arg Le - #u Leu Glu Ser Val                          350  - #               355  - #               360              - - Gly Leu Lys Pro Ile Val Pro Asp Gly Gly Ty - #r Phe Ile Ile Ala                          365  - #               370  - #               375              - - Asp Val Ser Leu Leu Asp Pro Asp Leu Ser As - #p Met Lys Asn Asn                          380  - #               385  - #               390              - - Glu Pro Tyr Asp Tyr Lys Phe Val Lys Trp Me - #t Thr Lys His Lys                          395  - #               400  - #               405              - - Lys Leu Ser Ala Ile Pro Val Ser Ala Phe Cy - #s Asn Ser Glu Thr                          410  - #               415  - #               420              - - Lys Ser Gln Phe Glu Lys Phe Val Arg Phe Cy - #s Phe Ile Lys Lys                          425  - #               430  - #               435              - - Asp Ser Thr Leu Asp Ala Ala Glu Glu Ile Il - #e Lys Ala Trp Ser                          440  - #               445  - #               450              - - Val Gln Lys Ser                                                           - -  - - (2) INFORMATION FOR SEQ ID NO: 2:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 425 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAINOT09                                                        (B) CLONE: 2150892                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #2:                           - - Met Asn Tyr Ala Arg Phe Ile Thr Ala Ala Se - #r Ala Arg Arg Asn                            - #5                  - #10                  - #15           - - Pro Thr Pro Ile Arg Thr Met Thr Asp Ile Le - #u Ser Arg Gly Pro                           20 - #                 25 - #                 30              - - Lys Ser Met Ile Ser Leu Ala Gly Gly Leu Pr - #o Asn Pro Asn Met                           35 - #                 40 - #                 45              - - Phe Pro Phe Lys Thr Ala Val Ile Thr Val Gl - #u Asn Gly Lys Thr                           50 - #                 55 - #                 60              - - Ile Gln Phe Gly Glu Glu Met Met Lys Arg Al - #a Leu Gln Tyr Ser                           65 - #                 70 - #                 75              - - Pro Ser Ala Gly Ile Pro Glu Leu Leu Ser Tr - #p Leu Lys Gln Leu                           80 - #                 85 - #                 90              - - Gln Ile Lys Leu His Asn Pro Pro Thr Ile Hi - #s Tyr Pro Pro Ser                           95 - #                100 - #                105              - - Gln Gly Gln Met Asp Leu Cys Val Thr Ser Gl - #y Ser Gln Gln Gly                          110  - #               115  - #               120              - - Leu Cys Lys Val Phe Glu Met Ile Ile Asn Pr - #o Gly Asp Asn Val                          125  - #               130  - #               135              - - Leu Leu Asp Glu Pro Ala Tyr Ser Gly Thr Le - #u Gln Ser Leu His                          140  - #               145  - #               150              - - Pro Leu Gly Cys Asn Ile Ile Asn Val Ala Se - #r Asp Glu Ser Gly                          155  - #               160  - #               165              - - Ile Val Pro Asp Ser Leu Arg Asp Ile Leu Se - #r Arg Trp Lys Pro                          170  - #               175  - #               180              - - Glu Asp Ala Lys Asn Pro Gln Lys Asn Thr Pr - #o Lys Phe Leu Tyr                          185  - #               190  - #               195              - - Thr Val Pro Asn Gly Asn Asn Pro Thr Gly As - #n Ser Leu Thr Ser                          200  - #               205  - #               210              - - Glu Arg Lys Lys Glu Ile Tyr Glu Leu Ala Ar - #g Lys Tyr Asp Phe                          215  - #               220  - #               225              - - Leu Ile Ile Glu Asp Asp Pro Tyr Tyr Phe Le - #u Gln Phe Asn Lys                          230  - #               235  - #               240              - - Phe Arg Val Pro Thr Phe Leu Ser Met Asp Va - #l Asp Gly Arg Val                          245  - #               250  - #               255              - - Ile Arg Ala Asp Ser Phe Ser Lys Ile Ile Se - #r Ser Gly Leu Arg                          260  - #               265  - #               270              - - Ile Gly Phe Leu Thr Gly Pro Lys Pro Leu Il - #e Glu Arg Val Ile                          275  - #               280  - #               285              - - Leu His Ile Gln Val Ser Thr Leu His Pro Se - #r Thr Phe Asn Gln                          290  - #               295  - #               300              - - Leu Met Ile Ser Gln Leu Leu His Glu Trp Gl - #y Gly Glu Gly Phe                          305  - #               310  - #               315              - - Met Ala His Val Asp Arg Val Ile Asp Phe Ty - #r Ser Asn Gln Lys                          320  - #               325  - #               330              - - Asp Ala Ile Leu Ala Ala Ala Asp Lys Trp Le - #u Thr Gly Leu Ala                          335  - #               340  - #               345              - - Glu Trp His Val Pro Ala Ala Gly Met Phe Le - #u Trp Ile Lys Val                          350  - #               355  - #               360              - - Lys Gly Ile Asn Asp Val Lys Glu Leu Ile Gl - #u Glu Lys Ala Val                          365  - #               370  - #               375              - - Lys Met Gly Val Leu Met Leu Pro Gly Asn Al - #a Phe Tyr Val Asp                          380  - #               385  - #               390              - - Ser Ser Ala Pro Ser Pro Tyr Leu Arg Ala Se - #r Phe Ser Ser Ala                          395  - #               400  - #               405              - - Ser Pro Glu Gln Met Asp Val Ala Phe Gln Va - #l Leu Ala Gln Leu                          410  - #               415  - #               420              - - Ile Lys Glu Ser Leu                                                                      425                                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 3:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 447 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAITUT21                                                        (B) CLONE: 2525071                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #3:                           - - Met Met Gln Asp Tyr Val Arg Thr Gly Thr Ty - #r Gln Arg Ala Ile                            - #5                  - #10                  - #15           - - Leu Gln Asn His Thr Asp Phe Lys Asp Lys Il - #e Val Leu Asp Val                           20 - #                 25 - #                 30              - - Gly Cys Gly Ser Gly Ile Leu Ser Phe Phe Al - #a Ala Gln Ala Gly                           35 - #                 40 - #                 45              - - Ala Arg Lys Ile Tyr Ala Val Glu Ala Ser Th - #r Met Ala Gln His                           50 - #                 55 - #                 60              - - Ala Glu Val Leu Val Lys Ser Asn Asn Leu Th - #r Asp Arg Ile Val                           65 - #                 70 - #                 75              - - Val Ile Pro Gly Lys Val Glu Glu Val Ser Le - #u Pro Glu Gln Val                           80 - #                 85 - #                 90              - - Asp Ile Ile Ile Ser Glu Pro Met Gly Tyr Me - #t Leu Phe Asn Glu                           95 - #                100 - #                105              - - Arg Met Leu Glu Ser Tyr Leu His Ala Lys Ly - #s Tyr Leu Lys Pro                          110  - #               115  - #               120              - - Ser Gly Asn Met Phe Pro Thr Ile Gly Asp Va - #l His Leu Ala Pro                          125  - #               130  - #               135              - - Phe Thr Asp Glu Gln Leu Tyr Met Glu Gln Ph - #e Thr Lys Ala Asn                          140  - #               145  - #               150              - - Phe Trp Tyr Gln Pro Ser Phe His Gly Val As - #p Leu Ser Ala Leu                          155  - #               160  - #               165              - - Arg Gly Ala Ala Val Asp Glu Tyr Phe Arg Gl - #n Pro Val Val Asp                          170  - #               175  - #               180              - - Thr Phe Asp Ile Arg Ile Leu Met Ala Lys Se - #r Val Lys Tyr Thr                          185  - #               190  - #               195              - - Val Asn Phe Leu Glu Ala Lys Glu Gly Asp Le - #u His Arg Ile Glu                          200  - #               205  - #               210              - - Ile Pro Phe Lys Phe His Met Leu His Ser Gl - #y Leu Val His Gly                          215  - #               220  - #               225              - - Leu Ala Phe Trp Phe Asp Val Ala Phe Ile Gl - #y Ser Ile Met Thr                          230  - #               235  - #               240              - - Val Trp Leu Ser Thr Ala Pro Thr Glu Pro Le - #u Thr His Trp Tyr                          245  - #               250  - #               255              - - Gln Val Arg Cys Leu Phe Gln Ser Pro Leu Ph - #e Ala Lys Ala Gly                          260  - #               265  - #               270              - - Asp Thr Leu Ser Gly Thr Cys Leu Leu Ile Al - #a Asn Lys Arg Gln                          275  - #               280  - #               285              - - Ser Tyr Asp Ile Ser Ile Val Ala Gln Val As - #p Gln Thr Gly Ser                          290  - #               295  - #               300              - - Lys Ser Ser Asn Leu Leu Asp Leu Lys Asn Pr - #o Phe Phe Arg Tyr                          305  - #               310  - #               315              - - Thr Gly Thr Thr Pro Ser Pro Pro Pro Gly Se - #r His Tyr Thr Ser                          320  - #               325  - #               330              - - Pro Ser Glu Asn Met Trp Asn Thr Gly Ser Th - #r Tyr Asn Leu Ser                          335  - #               340  - #               345              - - Ser Gly Met Ala Val Ala Gly Met Pro Thr Al - #a Tyr Asp Leu Ser                          350  - #               355  - #               360              - - Ser Val Ile Ala Ser Gly Ser Ser Val Gly Hi - #s Asn Asn Leu Ile                          365  - #               370  - #               375              - - Pro Leu Ala Asn Thr Gly Ile Val Asn His Th - #r His Ser Arg Met                          380  - #               385  - #               390              - - Gly Ser Ile Met Ser Thr Gly Ile Val Gln Gl - #y Ser Ser Gly Ala                          395  - #               400  - #               405              - - Gln Gly Ser Gly Gly Gly Ser Thr Ser Ala Hi - #s Tyr Ala Val Asn                          410  - #               415  - #               420              - - Ser Gln Phe Thr Met Gly Gly Pro Ala Ile Se - #r Met Ala Ser Pro                          425  - #               430  - #               435              - - Met Ser Ile Pro Thr Asn Thr Met His Tyr Gl - #y Ser                                      440  - #               445                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 4:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 1975 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PROSNOT20                                                        (B) CLONE: 1815528                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #4:                           - - CAAAAAAACA CCCCTCATCC AGTCTCTTCC AGCCTAGAGA TCCTGGCCTA CC -             #CCTCCGCC     60                                                                 - - AAAGCGCGCA CTGAGTGCAA ACCCCAGAGT CAATCCCTGT CCCGGCTCCG CC -            #CCCCGCGT    120                                                                 - - CCGAATCCCG CCCAGCCGGG CCCTCAAGCC CAGTCGGGAC TCGAGCCTAG GG -            #AGGCGAGG    180                                                                 - - TTCCCGCACC GGATAGCATG TTTTTGGCCC AGAGGAGCCT CTGCTCTCTT AG -            #CGGTAGAG    240                                                                 - - CAAAATTCCT GAAGACAATT TCTTCTTCCA AAATCCTCGG ATTCTCTACT TC -            #TGCTAAAA    300                                                                 - - TGTCACTGAA ATTCACAAAT GCAAAACGGA TTGAAGGACT TGATAGTAAT GT -            #GTGGATTG    360                                                                 - - AATTTACCAA ATTGGCTGCA GACCCTTCTG TTGTGAATCT TGGCCAAGGC TT -            #TCCAGATA    420                                                                 - - TATCCCCTCC TACATATGTA AAAGAAGAAT TATCAAAGAT TGCAGCAATC GA -            #TAGCCTGA    480                                                                 - - ATCAGTATAC ACGAGGCTTT GGCCATCCAT CACTTGTGAA AGCTCTGTCC TA -            #TCTGTATG    540                                                                 - - AAAAGCTTTA TCAAAAGCAA ATTGATTCAA ATAAAGAAAT CCTTGTGACA GT -            #AGGAGCAT    600                                                                 - - ATGGATCTCT TTTTAACACC ATTCAAGCAT TAATTGATGA GGGAGATGAA GT -            #CATACTAA    660                                                                 - - TAGTGCCTTT CTATGACTGC TATGAGCCCA TGGTGAGAAT GGCTGGAGCA AC -            #ACCTGTTT    720                                                                 - - TTATTCCCCT GAGATCTAAA CCTGTTTATG GAAAAAGATG GTCTAGTTCT GA -            #CTGGACAT    780                                                                 - - TAGATCCTCA AGAACTGGAA AGTAAATTTA ATTCCAAAAC CAAAGCTATT AT -            #ACTAAATA    840                                                                 - - CTCCACATAA CCCACTTGGC AAGGTGTATA ACAGAGAGGA ACTGCAAGTA AT -            #TGCTGACC    900                                                                 - - TTTGCATCAA ATATGACACA CTCTGCATCA GTGATGAGGT TTATGAATGG CT -            #TGTATATT    960                                                                 - - CTGGAAATAA GCACTTAAAA ATAGCTACTT TTCCAGGTAT GTGGGAGAGA AC -            #AATAACAA   1020                                                                 - - TAGGAAGTGC TGGAAAGACT TTCAGTGTAA CTGGCTGGAA GCTTGGCTGG TC -            #CATTGGTC   1080                                                                 - - CAAATCATTT GATAAAACAT TTACAGACAG TTCAACAAAA CACGATTTAT AC -            #TTGTGCAA   1140                                                                 - - CTCCTTTACA GGAAGCCTTG GCTCAAGCTT TCTGGATTGA CATCAAGCGC AT -            #GGATGACC   1200                                                                 - - CAGAATGTTA CTTTAATTCT TTGCCAAAAG AGTTAGAAGT AAAAAGAGAT CG -            #GATGGTAC   1260                                                                 - - GTTTACTTGA AAGTGTTGGC CTAAAACCCA TAGTTCCTGA TGGAGGATAC TT -            #CATCATCG   1320                                                                 - - CTGATGTGTC TTTGCTAGAT CCAGACCTCT CTGATATGAA GAATAATGAG CC -            #TTATGACT   1380                                                                 - - ATAAGTTTGT GAAATGGATG ACTAAACATA AGAAACTATC AGCCATCCCC GT -            #TTCAGCAT   1440                                                                 - - TCTGTAACTC AGAGACTAAA TCACAGTTTG AGAAGTTTGT GCGTTTTTGC TT -            #CATTAAAA   1500                                                                 - - AAGACAGCAC ACTGGATGCT GCTGAAGAAA TCATCAAGGC ATGGAGTGTA CA -            #GAAGTCTT   1560                                                                 - - GATTTGTGCA GAATGGATTA ATGTTTCTGT TAGATGACCT AGTATGGAAT TG -            #TTACTTAG   1620                                                                 - - TGCTGCCACC TGCTGGATGT TAAAAGGTAT TTCAGTACAA CTGGAATTTA AA -            #TATTTCCA   1680                                                                 - - TTGTTTTTCC AAAGCAGTTA ACCCAACTCC TAACAACATT TTCGGGGGAT CT -            #GACCTTTT   1740                                                                 - - TTTTCCAGTT GAAATGTATT AACACACCTT CCACAATCAT TTTATAAGAG TC -            #AGCATAAC   1800                                                                 - - ATAGTGGATA AGAACTGTGA GATGTTTAAC CTCTCAGTAA CTCGGTTCTC TC -            #ATTATAAA   1860                                                                 - - ATAGGAATAA AATCAGTACC TGTTTCATAT GAAGGTCGTT TCTGAGAATT AA -            #ATGGACTA   1920                                                                 - - ATGTATGCAA AAAGCCTGGC AAACAATAAA CACTCATCTG ACTTTAAAAA AA - #AAA            1975                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 5:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2125 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAINOT09                                                        (B) CLONE: 2150892                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #5:                           - - GCGCGTGGGA AGCCAGACGC AGCGGGGGGA CACATCTCGC GGTGGCGTTG CC -             #AGAGTGAG     60                                                                 - - GAGTTAGCAG GCAGGACTTG ACGAGGCTCT TTGGTTTTTC TAGTCCTCAA CC -            #ACTGAAGA    120                                                                 - - AGAAGCTTGA TGCTTGGCTG TCAGAAGACA TGAATTACGC ACGGTTCATC AC -            #GGCAGCGA    180                                                                 - - GCGCACGCAG AAACCCTACT CCCATCCGGA CCATGACTGA CATATTGAGC AG -            #AGGACCAA    240                                                                 - - AATCGATGAT CTCCTTGGCT GGTGGCTTAC CAAATCCAAA CATGTTTCCT TT -            #TAAGACTG    300                                                                 - - CCGTAATCAC TGTAGAAAAT GGAAAGACCA TCCAATTTGG AGAAGAGATG AT -            #GAAGAGAG    360                                                                 - - CACTTCAGTA TTCTCCGAGT GCTGGAATTC CAGAGCTTTT GTCCTGGCTA AA -            #ACAGTTAC    420                                                                 - - AAATAAAATT GCATAATCCT CCTACCATCC ATTACCCACC CAGTCAAGGA CA -            #AATGGATC    480                                                                 - - TATGTGTCAC ATCTGGCAGC CAACAAGGTC TTTGTAAGGT GTTTGAAATG AT -            #CATTAATC    540                                                                 - - CTGGAGATAA TGTCCTCCTA GATGAACCTG CTTATTCAGG AACTCTTCAA AG -            #TCTGCACC    600                                                                 - - CACTGGGCTG CAACATTATT AATGTTGCCA GTGATGAGAG TGGGATTGTT CC -            #AGATTCCC    660                                                                 - - TAAGAGACAT ACTTTCCAGA TGGAAACCAG AAGATGCAAA GAATCCCCAG AA -            #AAACACCC    720                                                                 - - CCAAATTTCT TTATACTGTT CCAAATGGCA ACAACCCTAC TGGAAACTCA TT -            #AACCAGTG    780                                                                 - - AACGCAAAAA GGAAATCTAT GAGCTTGCAA GAAAATATGA TTTCCTCATA AT -            #AGAAGATG    840                                                                 - - ATCCTTACTA TTTTCTCCAG TTTAACAAGT TCAGGGTACC AACATTTCTT TC -            #CATGGATG    900                                                                 - - TTGATGGACG TGTCATCAGA GCTGACTCTT TTTCAAAAAT CATTTCCTCT GG -            #GTTGAGAA    960                                                                 - - TAGGATTTTT AACTGGTCCA AAACCCTTAA TAGAGAGAGT TATTTTACAC AT -            #ACAAGTTT   1020                                                                 - - CAACATTGCA CCCCAGCACT TTTAACCAGC TCATGATATC ACAGCTTCTA CA -            #CGAATGGG   1080                                                                 - - GAGGAGAAGG TTTCATGGCT CATGTAGACA GGGTTATTGA TTTCTATAGT AA -            #CCAGAAGG   1140                                                                 - - ATGCAATACT GGCAGCTGCA GACAAGTGGT TAACTGGTTT GGCAGAATGG CA -            #TGTTCCTG   1200                                                                 - - CTGCTGGAAT GTTTTTATGG ATTAAAGTTA AAGGCATTAA TGATGTAAAA GA -            #ACTGATTG   1260                                                                 - - AAGAAAAGGC CGTTAAGATG GGGGTATTAA TGCTCCCTGG AAATGCTTTC TA -            #CGTCGATA   1320                                                                 - - GCTCAGCTCC TAGCCCTTAC TTGAGAGCAT CCTTCTCTTC AGCTTCTCCA GA -            #ACAGATGG   1380                                                                 - - ATGTGGCCTT CCAGGTATTA GCACAACTTA TAAAAGAATC TTTATGAAGA AA -            #TTAAACTA   1440                                                                 - - GGTTGGGCAT GGTGGCTCAC ACCTATAATC CCAGCACTTT GGGAGGCAGA GG -            #AGGGAGGA   1500                                                                 - - TCACTTGGAC CCAGGAATTC AAGGCTGCAG TAAGCTACGA TCACACCACT GC -            #ACTCTGGC   1560                                                                 - - CTGCATGCAC TCTGGCCTGC ATGGCAGAAC AAGACCCTGT CTCTAAAAAA AG -            #AGAAAGAA   1620                                                                 - - ATCAAACTAA TCATGCTGCT CATGGATTTT TCCAATAAAT TTCTTGTTTT GG -            #CAGGAAGA   1680                                                                 - - AATGAACACT GGTATTAGAC TTAAAGATTA AATTTCCTCA AACATGTCCT AT -            #CTGTAGTA   1740                                                                 - - GTTCAACTAG ACACCTTTTA AAGTGCCTCT AAATTCATCA GATGGCCAAA CT -            #GTATTTAT   1800                                                                 - - AATCCACTTA GGCATTTTGA AAAACTTTCA ACCTGTAAAA AGTTACTTTT AT -            #CTTGGATT   1860                                                                 - - TATTATGAAG AACTTTGTAG TTGCTTTGTA ATTTCCCATA AATTGTCTTT GA -            #AACTAACA   1920                                                                 - - TTTTACACTG AATTATTTTG AGATTTTAAA GAAGTAATTA AGTGCAAAAT GG -            #TATATAAT   1980                                                                 - - GTGTACTTTT TCTACTTTTA GGAAAATTTA ATGAGAGCTT ATTGCAAAAA TT -            #GTTATAAT   2040                                                                 - - TTGGTCATTA TAAGTGACTT TTAGTAAAAG TACCATAAAC CTTATGTTAT GC -            #CACAGAAA   2100                                                                 - - TTCCTTTAAA ATAAAATTCT TAAAT          - #                  - #                 2125                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 6:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 2224 base - #pairs                                                (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAITUT21                                                        (B) CLONE: 2525071                                                   - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #6:                           - - CAGAGTGCAG CCGTGTGGGC AAGCAGTCCT TCATCATCAC CCTGGGCTGC AA -             #CAGCGTCC     60                                                                 - - TCATCCAGTT CGCCACACCC AACGATTCGG CTCGAGCTTC TACAACATCC TG -            #AAAACCTG    120                                                                 - - CCGGGGCCAC ACCCTGGAGC GGTCTGTGTT CAGCGAGCGG ACGGAGGAGT CT -            #TCTGCCGT    180                                                                 - - GCAGTACTTC CAGTTTTATG GCTACCTGTC CCAGCAGCAG AACATGATGC AG -            #GACTACGT    240                                                                 - - GCGGACAGGC ACCTACCAGC GCGCCATCCT GCAAAACCAC ACCGACTTCA AG -            #GACAAGAT    300                                                                 - - CGTTCTTGAT GTTGGCTGTG GCTCTGGGAT CCTGTCGTTT TTTGCCGCCC AA -            #GCTGGAGC    360                                                                 - - ACGGAAAATC TACGCGGTGG AGGCCAGCAC CATGGCCCAG CACGCTGAGG TC -            #TTGGTGAA    420                                                                 - - GAGTAACAAC CTGACGGACC GCATCGTGGT CATCCCGGGC AAGGTGGAGG AG -            #GTGTCACT    480                                                                 - - CCCCGAGCAG GTGGACATCA TCATCTCGGA GCCCATGGGC TACATGCTCT TC -            #AACGAGCG    540                                                                 - - CATGCTGGAG AGCTACCTCC ACGCCAAGAA GTACCTGAAG CCCAGCGGAA AC -            #ATGTTTCC    600                                                                 - - TACCATTGGT GACGTCCACC TTGCACCCTT CACGGATGAA CAGCTCTACA TG -            #GAGCAGTT    660                                                                 - - CACCAAGGCC AACTTCTGGT ACCAGCCATC TTTCCATGGA GTGGACCTGT CG -            #GCCCTCCG    720                                                                 - - AGGTGCCGCG GTGGATGAGT ATTTCCGGCA GCCTGTGGTG GACACATTTG AC -            #ATCCGGAT    780                                                                 - - CCTGATGGCC AAGTCTGTCA AGTACACGGT GAACTTCTTA GAAGCCAAAG AA -            #GGAGATTT    840                                                                 - - GCACAGGATA GAAATCCCAT TCAAATTCCA CATGCTGCAT TCAGGGCTGG TC -            #CACGGCCT    900                                                                 - - GGCTTTCTGG TTTGACGTTG CTTTCATCGG CTCCATAATG ACCGTGTGGC TG -            #TCCACAGC    960                                                                 - - CCCGACAGAG CCCCTGACCC ACTGGTACCA GGTGCGGTGC CTGTTCCAGT CA -            #CCACTGTT   1020                                                                 - - CGCCAAGGCA GGGGACACGC TCTCAGGGAC ATGTCTGCTT ATTGCCAACA AA -            #AGACAGAG   1080                                                                 - - CTACGACATC AGTATTGTGG CCCAGGTGGA CCAGACCGGC TCCAAGTCCA GT -            #AACCTCCT   1140                                                                 - - GGATCTGAAA AACCCCTTCT TTAGATACAC GGGCACAACG CCCTCACCCC CA -            #CCCGGCTC   1200                                                                 - - CCACTACACA TCTCCCTCGG AAAACATGTG GAACACGGGC AGCACCTACA AC -            #CTCAGCAG   1260                                                                 - - CGGGATGGCC GTGGCAGGGA TGCCGACCGC CTATGACTTG AGCAGTGTTA TT -            #GCCAGTGG   1320                                                                 - - CTCCAGCGTG GGCCACAACA ACCTGATTCC TTTAGCCAAC ACGGGGATTG TC -            #AATCACAC   1380                                                                 - - CCACTCCCGG ATGGGCTCCA TAATGAGCAC GGGGATTGTC CAAGGGTCCT CC -            #GGCGCCCA   1440                                                                 - - GGGCAGTGGT GGTGGCAGCA CGAGTGCCCA CTATGCAGTC AACAGCCAGT TC -            #ACCATGGG   1500                                                                 - - CGGCCCCGCC ATCTCCATGG CGTCGCCCAT GTCCATCCCG ACCAACACCA TG -            #CACTACGG   1560                                                                 - - GAGCTAGGGG CCCGCCCCGC GGACTGACAG CACCAGGAAA CCAAATGATG TC -            #CCTGCCCG   1620                                                                 - - CCGCCCCCGC CGGGCGGCTT TCCCCCTTGT ACTGGAGAAG CTCGAACACC CG -            #GTCACAGC   1680                                                                 - - TCTCTTTGCT ATGGGAACTG GGACACTTTT TTACACGATG TTGCCGCCGT CC -            #CCACCCTA   1740                                                                 - - ACCCCCACCT CCCGGCCCTG AGCGTGTGTC GCTGCCATAT TTTACACAAA AT -            #CATGTTGT   1800                                                                 - - GGGAGCCCTC GTCCCCCCTC CTGCCCGCTC TACCCTGACC TGGGCTTGTC AT -            #CTGCTGGA   1860                                                                 - - ACAGGCGCCA TGGGGCCTGC CAGCCCTGCC TGCCAGGTCC CTTAGCACCT GT -            #CCCCCTGC   1920                                                                 - - CTGTCTCCAG TGGGAAGGTA GCCTGGCCAG GCGGGGCCTC CCCTTCGACG AC -            #CAGGCCTC   1980                                                                 - - GGTCACAACG GACGTGACAT GCTGCTTTTT TTAATTTTAT TTTTTTATGA AA -            #AGAACCAG   2040                                                                 - - TGTCAATCCG CAGACCCTCT GTGAAGCCAG GCCGGCCGGG CCGAGCCAGC AG -            #CCCCTCTC   2100                                                                 - - CCTAGACTCA GAGGCGCCGC GGGGAGGGGT GGCCCCGCCG AGGCTTCAGG GG -            #CCCCCTCC   2160                                                                 - - CCACCAAAGG GTTCACCTCA CACTTGAATG TACAACCCAC CCCACTGTCG GG -            #AAGGCCTC   2220                                                                 - - CGTC                 - #                  - #                  - #               2224                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 7:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 244 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PROSNOT20                                                        (B) CLONE: 1815528H1                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #7:                           - - ATTATACTAA ATACTCCACA TAACCCACTT GGCAAGGTGT ATAACAGAGA GG -             #AACTGCAA     60                                                                 - - GTAATTGCTG ACCTTTGCAT CAAATATGAC ACACTCTGCA TCAGTGATGA GG -            #TTTATGAA    120                                                                 - - TGGCTTGTAT ATTCTGGAAA TAAGCACTTA AAAATAGCTA CTTTTCCAGG TA -            #TGTGGGAG    180                                                                 - - AGAACAATAA CAATAGGAAG TGCTGGAAAG ACTTTCAGTG TAACTGGCTG GA -            #AGCTTGGC    240                                                                 - - TGGT                 - #                  - #                  - #                244                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 8:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 528 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: UTRSTUT05                                                        (B) CLONE: 2880980F6                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #8:                           - - ATTAAAGTTA CATTATCTAA AAAAAAAACT AGAAATAACT ATACTGGCTA AA -             #TTATAACA     60                                                                 - - CACTTATTTT CATTGAATTT ATGTATCTTT GTTATGTTTT TAGATTATAC TA -            #AATGTGAT    120                                                                 - - TAATTGGAAA ACAATATTTA CCCTTTTTTC CTCCTCTGTT TAGCTACTTT TC -            #CAGGTATG    180                                                                 - - TGGGAGAGAA CAATAACAAT AGGAAGTGCT GGAAAGACTT TCAGTGTAAC TG -            #GCTGGAAG    240                                                                 - - CTTGGCTGGT CCATTGGTCC AAATCATTTG ATAAAACATT TACAGACAGT TC -            #AACAAAAC    300                                                                 - - ACGATTTATA CTTGTGCAAC TCCTTTACAG GAAGCCTTGG CTCAAGCTTT CT -            #GGATTGAC    360                                                                 - - ATCAAGCGCA TGGATGACCC AGAATGTTAC TTTAATTCTT TGCCAAAAGN GT -            #TAGAAGTA    420                                                                 - - AAAAGAGATC GGATGGTACG TTTACTTGAA AAGTGTTGGG CCTAAAAACC CA -            #TAGTTCCN    480                                                                 - - GGANGGAGGG ATACTTCATC ATCGGCTGGA TGNGGNCTTT GGCCAGAT  - #                   528                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 9:                                    - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 622 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PROSNOT20                                                        (B) CLONE: 1815528X12C1                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #9:                           - - CAATCGATAG CCTGNAATCA GTATACACGA GGCTTTGGCC ATCCATCACT TG -             #TGAAAGCT     60                                                                 - - CTGTCCTATC TGTATGAAAA GCTTTATCAA AAGCAAATTG ATTCAAATAA AG -            #AAATCCTT    120                                                                 - - GTGACAGTAG GAGCATATGG ATCTCTTTTT AACACCATTC AAGCATTAAT TG -            #ATGAGGGA    180                                                                 - - GATGAAGTCA TACTAATAGT GCCTTTCTAT GACTGCTATG AGCCCATGGT GA -            #GAATGGCT    240                                                                 - - GGAGCAACAC CTGTTTTTAT TCCCCTGAGA TCTAAACCTG TTTATGGAAA AA -            #GATGGTCT    300                                                                 - - AGTTCTGACT GGACATTAGA TCCTCAAGAA CTGGAAAGTA AATTTAATTC CA -            #AAACCAAA    360                                                                 - - GCTATTATAC TAAATACTCC ACATAACCCA CTTGGCAAGG TGTATAACAG AG -            #AGGAACTG    420                                                                 - - CAAGTAATTG CTGACCTTTG CATCAAATAT GACACACTCT GATTCAGTGA TG -            #AGGTTTAT    480                                                                 - - GAATGGCTTG TATATTCGGA AATAAGCACT AAAAATAGCT ACTTTCCGGT AT -            #GTGGGAGA    540                                                                 - - GAACAATAAC AATAGGAAGT GCTGGAAAGA CTTCGTGTAA CTGGCTGGAA GC -            #TGGGCTGG    600                                                                 - - TCCTTNGTCC AATCATTGAT AA           - #                  - #                    622                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 10:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 602 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PROSNOT20                                                        (B) CLONE: 1815528X17C1                                              - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #10:                          - - AGCATTAATT GATGAGGGAG ATGAAGTCAT ACTAATAGTG CCTTTCTATG AC -             #TGCTATGA     60                                                                 - - GCCCATGGTG AGAATGGCTG GAGCAACACC TGTTTTTATT CCCCTGAGAT CT -            #AAACCTGT    120                                                                 - - TTATGGAAAA AGATGGTCTA GTTCTGACTG GACATTAGAT CCTCAAGAAC TG -            #GAAAGTAA    180                                                                 - - ATTTAATTCC AAAACCAAAG CTATTATACT AAATACTCCA CATAACCCAC TT -            #GGCAAGGT    240                                                                 - - GTATAACAGA GAGGAACTGC AAGTAATTGC TGACCTTTGC ATCAAATATG AC -            #ACACTCTG    300                                                                 - - CATCAGTGAT GAGGTTTATG AATGGCTTGT ATATTCTGGA AATAAGCACT TA -            #AAAATAGC    360                                                                 - - TACTTTTCCA GGTATGTGGG AGAGAACAAT AACAATAGGA AGTGCTGGAA AG -            #ACTTTCAG    420                                                                 - - TGTAACTGGG CTGGAAGCTT GGCTGGTCCA TTGGTTCCAA ATTCTTTGAT AA -            #AACTTTAC    480                                                                 - - AGACGTTCAA CAAAACACGA TTTATACTGT GGCAACTTCC TTTACAGGAA GC -            #CTGGCTCA    540                                                                 - - AGCTTCTGGA TTGACTCAAG CGCTGGATGA CCCGAATGTT ACTTAATTCT TG -            #CCAAAGAG    600                                                                 - - TA                  - #                  - #                  - #                 602                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 11:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 789 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: PROSNOT20                                                        (B) CLONE: 1819092T6                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #11:                          - - CNAATTTTTA ATTTAATATT TGGTAAGTGT GTGGTTATAT AATTATTTTC AT -             #CATAAGAA     60                                                                 - - TTATGGANAA AATTTACAAA TNACAAAAAT TTGTGATAAT TTACCTTTTT CT -            #CATACTAA    120                                                                 - - ATCCTTCAAG GCCTCACTGT CTTTTTCCTG GGGTCCTGGA GCCTTCTGAC TG -            #GCTTCTCT    180                                                                 - - CTGTGCATTG CAGGACTTCT CCTCAGGTCA ACTTATCTTC TTNTAACATC CA -            #NCAGGTNG    240                                                                 - - CAGCNCTAAN TAACNANTCC ATACTAGGTC ATCTAACAGA AACATNAATC CA -            #TTCTGCAN    300                                                                 - - NANTCAAGAC TTCTGTACAC TCCANGCCTN GATGATTTCT TCAGCANCAT CC -            #AGTGTGCT    360                                                                 - - GNCTTTTTTA ATGAAGCANA AACGCNCAAA CTTCTCAAAC TGTGATTTAG TC -            #TCTGAGTT    420                                                                 - - ACAGAATGCT GAAACGGGGA TGGCTGATAG TTTCTTATGN TTAGTCATCC AN -            #TTNACNAA    480                                                                 - - CTTATNGTCA TAAGGCTCAT TANTCTCATA TCAGAGATGG CTGGATCTAG CA -            #AAGTCACA    540                                                                 - - TCAGCGATGA TGAAGTATCC TCCNACAGGA CTATGGGGTT TAGGCCACAC TN -            #TCAAGTAN    600                                                                 - - ACGTACCNTC CGNGTCTNCT TACTCCAACT CTTTNGNAAG ATTAAAGTAA CA -            #TTCTGGGN    660                                                                 - - CAACATGCGC CGATGCATCN AGAAGCTNAN CCAGGGNCTG NTNNAGATCT AA -            #AACCGAAA    720                                                                 - - GNATTGGNCG TATTTNGTAC TTNNGTAGAA TGGAGCCGGT AATTTGGAAG AG -            #CNCCCGAA    780                                                                 - - ACGGATCTG                - #                  - #                      - #        789                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 12:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 646 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: HNT2NOT01                                                        (B) CLONE: 269916F1                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #12:                          - - AAAGTCAGAT GAGTGTTTAT TGTTTGCCAG GNTTTTTGCA TACATTAGTC CA -             #TTTAATTC     60                                                                 - - TCAGAAACGA CCTTCATATG AAACAGGTAC TGATTTTATT CCTATTTTAT AA -            #TGAGAGAA    120                                                                 - - CCGAGTTACT GAGAGGTTAA ACATCTCACA TTTCTTATCC ACTATGTTAT GC -            #TGACTCTT    180                                                                 - - ATAAAATGAT TGTGGAAGGT GTGTTAATAC ATTTCAACTG GAAAAAAAAG GT -            #CAGATCCC    240                                                                 - - CCGAAAATGT TGTTAGGAGT TGGGTTAACT GCTTTGGAAA AACAATGGAA AT -            #ATTTAAAT    300                                                                 - - TCCAGTTGTA CTGAAATACC TTTTAACATC CAGCAGGTGG CAGCACTAAG TA -            #ACAATTCC    360                                                                 - - ATACTAGGTC ATCTAACAGA AACATTAATC CATTCTGCAC AAATCAAGAC TT -            #CTGTACAC    420                                                                 - - TCCATGCCTT GATGATTTCT TCAGCAGCAT CCAGTGTGCT GTCTTTTTTA AT -            #GAAGCAAA    480                                                                 - - AACGCACAAA CTTCTCAACT GTGATTTAGT CTCTGAGTTA CAGANTGCTG AA -            #ACGGGGTG    540                                                                 - - GCTGNTAGTT TCTTATGTTT AGGCCATCCA TTTCACAAAC TTATAGNCAT AA -            #GGCTCATT    600                                                                 - - ATTCTTCATA TCAGNGNGGT CTGGGNCTGG CAANGGCACA TNAGCG   - #                    646                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 13:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 369 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: UCMCNOT02                                                        (B) CLONE: 1717401F6                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #13:                          - - AGCTAGTATG GAATTGTTAC TTAGTGCTGC CACCTGCTGG ATGTTAAAAG GN -             #ATTTCAGT     60                                                                 - - ACAACTGGAA TTTAAATATT TCCATTGTTT TTCCAAAGCA GTTAACCCAA CT -            #CCTAACAA    120                                                                 - - CATTTTCGGG GGATCTGACC TTTTTTTTCC AGTTGAAATG TATTAACACA CC -            #TTCCACAA    180                                                                 - - TCATTTTATA AGAGTCAGCA TAACATAGTG GATAAGAACT GTGAGATGTT TA -            #ACCTCTCA    240                                                                 - - GTAACTCGGT TCTCTCATTA TAAAATAGGA ATAAAATCAG TACCTGTTTC AT -            #ATGAAGGT    300                                                                 - - CGTTTCTGAG AATTAAATGG ACTAATGTAT GCAAAAAGCC TGGCAAACAA TA -            #AACACTCA    360                                                                 - - TCTGACTTT                - #                  - #                      - #        369                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 14:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 243 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAINOT09                                                        (B) CLONE: 2150892H1                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #14:                          - - GACCATCCAA TTTGGAGAAG AGATGATGAA GAGAGCACTT CAGTATTCTC CG -             #AGTGCTGG     60                                                                 - - AATTCCAGAG CTTTTGTCCT GGCTAAAACA GTTACAAATA AAATTGCATA AT -            #CCTCCTAC    120                                                                 - - CATCCATTAC CCACCCAGTC AAGGACAAAT GGATCTATGT GTCACATCTG GC -            #AGCCAACA    180                                                                 - - AGGTCTTTGT AAGGTGTTTG ANATGATCAT TAATCCTGGA GATAATGTCC TC -            #CTAGATGA    240                                                                 - - ACC                  - #                  - #                  - #                243                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 15:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 569 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: N/A                                                              (B) CLONE: SAGA00872F1                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #15:                          - - AGGCGCGTGG GAAGCCAGAC GCAGCGGGGG GACACATCTC GCGGTGGCGT TG -             #CAGAGTGA     60                                                                 - - GGNGTTAGCA GGCAGGACTT GACGAGGCTC TTTGGTTTTT CTAGTCCTCA AC -            #CACTGAAG    120                                                                 - - AAGAAGCTTG ATGCTTGGCT GTCAGAAGAC ATGAATTACG CACGGTTCAT CA -            #CGGCAGCG    180                                                                 - - AGCGCANCAG AAACCCTACT CCCATCCGGA CCATGACTGA CATATTGAGC AG -            #AGGACCAA    240                                                                 - - AATCGATGAT CTCCTTGGCT GGTGGCTTAC CAAATCCAAA CATGTTTCCT TT -            #TAAGACTG    300                                                                 - - CCGTAATCAC TGTAGAAAAT GGAAAGACCA TCCAATTTGG AGAAGAGATG AT -            #GAAGAGAG    360                                                                 - - CACTTCAGTA TTCTCCGAGT GCTGGAATTC CAGAGCTTTT GTCCTGGCTA AA -            #ACAGTTAC    420                                                                 - - AAATAAAATT GCATAATCCT CCTACCATCC ATTACCACCC AGTCAAGGAC AA -            #ATGGATCT    480                                                                 - - ATGTGTCACA TCTGGCAGCC AACAAGGTCT TTGTAAGGTG TTTGAAATGA TC -            #ATTAATCC    540                                                                 - - TGGAGATAAT GTCCTCCTAG ATGAACCTG         - #                  - #               569                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 16:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 526 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: N/A                                                              (B) CLONE: SAGA01877F1                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #16:                          - - GGTCGACTCT AGAGGATCCC CCCATGAAAC CTTCTCCTCC CCATTCGTGT AG -             #AAGCTGTG     60                                                                 - - ATATCATGAG CTGGTCAAAA GTGCTGGGGT GCAATGTTGA AACTTGTATG TG -            #TAAAATAA    120                                                                 - - CTCTCTCTAT TAAGGGTTTT GGACCAGTTA AAAATCCTAT TCTCAACCCA GA -            #GGGAATGA    180                                                                 - - TTTTTGAAAA AGAGTCAGCT CTGATGACAC GTCCATCAAC ATCCATGGAA AG -            #AAATGTTG    240                                                                 - - GTACCCTGAA CTTGTTAAAC TGGAGAAAAT AGTAAGGATC ATCTTCTATT AT -            #GAGGAAAT    300                                                                 - - CATATTTTCT TGCAAGCTCA TAGATTTCCT TTTTGCGTTC ACTGGTTAAT GA -            #GTTTCCAG    360                                                                 - - TAGGGTTGTT GCCATTTGGA ACAGTATAAA GAAATTTGGG GGTGTTTTTC TG -            #GGGATTCT    420                                                                 - - TTGCATCTTC TGGTTTCCAT CTGGAAAGTA TGTCTCTTAG GGAATCTGGA AC -            #AATCCCAC    480                                                                 - - TCTCATCACT GGCAACATTA ATAATGTTGC AGGGGTACCG AGCTCG   - #                    526                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 17:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 467 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: N/A                                                              (B) CLONE: SAGA01269R1                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #17:                          - - TTATTAATGT TGCCAGTNAT GAGAGTGGGA TTGTTCCAGA TTCCCTAAGA GA -             #CATACTTT     60                                                                 - - CCAGATGGAA ACCAGAAGAT GCAAAGAATC CCCAGAAAAA CACCCCCAAA TT -            #TCTTTATA    120                                                                 - - CTGTTCCAAA TGGCAACAAC CCTACTGGAA ACTCATTAAC CAGTGAACGC AA -            #AAAGGAAA    180                                                                 - - TCTATGAGCT TGCAAGAAAA TATGATTTCC TCATAATAGA AGATGATCCT TA -            #CTATTTTC    240                                                                 - - TCCAGTTTAA CAAGTTCAGG GTACCAACAT TTCTTTCCAT GGATGTTGAT GG -            #ACGTGTCA    300                                                                 - - TCAGAGCTGA CTCTTTTTCA AAAATCATTT CCTCTGGGTT GAGAATAGGA TT -            #TTTAACTG    360                                                                 - - GTCCAAAACC CTTAATAGAG AGAGTTATTT TACACATACA AGTTTCAACA TT -            #GCACCCCA    420                                                                 - - GCACTTTTAA CCAGCTCATG ATATCACAGG GGGATCCTCT AGAGTCG   - #                   467                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 18:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 338 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: N/A                                                              (B) CLONE: SAGA02228F1                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #18:                          - - GCAGGTCGAC TCTAGAGGAT CCCCCTGCAG ACAAGTGGTT AACTGGTTTG GC -             #AGAATGGC     60                                                                 - - ATGTTCCTGC TGCTGGAATG TTTTTATGGA TTAAAGTTAA AGGCATTAAT GA -            #TGTAAAAG    120                                                                 - - AACTGATTGA AGAAAAGGCC GTTAAGATGG GGGTATTAAT GCTCCCTGGA AA -            #TGCTTTCT    180                                                                 - - ACGTCGATAG CTCAGCTCCT AGCCCTTACT TGAGAGCATC CTTCTCTTCA GC -            #TTCTCCAG    240                                                                 - - AACAGATGGA TGTGGCCTTC CAGGTATTAG CACAACTTAT AAAAGAATCT TT -            #ATGAAGAA    300                                                                 - - ATTAAACTAG GTTGGGCATG GTGGGGGTAC CGAGCTCG      - #                      - #    338                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 19:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 605 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: N/A                                                              (B) CLONE: SAGA01614F1                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #19:                          - - TGCAGGTCGA CTCTAGAGGA TCCCCCCGTT AAGATGGGGG TATTAATGCT CC -             #CTGGAAAT     60                                                                 - - GCTTTCTACG TCGATAGCTC AGCTCCTAGC CCTTACTTGA GAGCATCCTT CT -            #CTTCAGCT    120                                                                 - - TCTCCAGAAC AGATGGATGT GGCCTTCCAG GTATTAGCAC AACTTATAAA AG -            #AATCTTTA    180                                                                 - - nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn -            #nnnnnnnn    240                                                                 - - nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn -            #nnnnnnnn    300                                                                 - - nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nn -            #nnnnnnnn    360                                                                 - - nnnnnnnnnn nnnnnnnnnn nnnnnnTCAT GCTGCTCATG GATTTTTCCA AT -            #AAATTTCT    420                                                                 - - TGTTTTGGCA GGAAGAAATG AACACTGGTA TTAGACTTAA AGATTAAATT TC -            #CTCAAACA    480                                                                 - - TGTCCTATTC TGTAGNAGTT CAACTAGACA CCTTTTAAAG TGCCTCTAAA TT -            #CATCAGAT    540                                                                 - - GGCCAAACTG TATTTATAAT CCACTTAGGC ATTTTGAAAA ACTTCAACCT GT -            #AAAAAGNT    600                                                                 - - ACTTT                 - #                  - #                  -      #           605                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 20:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 495 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: TESTNOT04                                                        (B) CLONE: 301251T6                                                  - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #20:                          - - GTTAAGAATT TTATTTTAAA GGAATTTCTG TGGCATAACA TAAGGTTTAT GG -            #TACTTTTA     60                                                                 - - CTAAAAGTCA CTTATAATGA CCAAATTATA ACAATTTTTG CAATAAGCTC TC -            #ATTAAATT    120                                                                 - - TTCCTAAAAG TAGAAAAAGT ACACATTATA TACCATTTTG CACTTAATTA CT -            #TCTTTAAA    180                                                                 - - ATCTCAAAAT AATTCAGTGT AAAATGTTAG TTTCAAAGAC AATTTATGGG AA -            #ATTACAAA    240                                                                 - - GCAACTACAA AGTTCTTCAT AATAANTCCA AGATAAAAGT AACTTTTTAC AG -            #GTTGAAAG    300                                                                 - - TTTTTCAAAA TGCCTAAGTG GATTATAAAT ACAGTTTGGC CATCTGATGA AT -            #TTAGAGGC    360                                                                 - - ACTTTAAAAG GTGTCTAGTT GAACTACTAC AGATAGGACA TGTTTGAGGA AA -            #TTTAATCT    420                                                                 - - TTAAGTCTAA TACCAGTGGT CATTTCCTCC TGCCAAAACA AGANATTTAT TG -            #GAAAAATC    480                                                                 - - CATGAGCAGC ATGAT              - #                  - #                      - #   495                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 21:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 256 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAITUT21                                                        (B) CLONE: 2525071H1                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #21:                          - - CTTCTACAAC ATCCTGAAAA CCTGCCGGGG CCACACCCTG GAGCGGTCTG TG -             #TTCAGCGA     60                                                                 - - GCGGACGGAG GAGTCTTCTG CCGTGCAGTA CTTCCAGTTT TATGGCTACC TG -            #TCCCAGCA    120                                                                 - - GCAGAACATG ATGCAGGACT ACGTGCGGAC AGGCACCTAC CAGCGCGCCA TC -            #CTGCAAAA    180                                                                 - - CCACACCGAC TTCAAGGACA AGATCGTTCT TGATGTTGGC TGTGGCTCTG GG -            #ATCCTGTC    240                                                                 - - GTTTTTTGCC GCCCAA             - #                  - #                      - #   256                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 22:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 258 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BLADTUT07                                                        (B) CLONE: 1889292H1                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #22:                          - - CAGAGTGCAG CCGTGTGGGC AAGCAGTCCT TCATCATCAC CCTGGGCTGC AA -             #CAGCGTCC     60                                                                 - - TCATCCAGTT CGCCACACCC AACGATTTCT GTTCCTTCTA CAACATCCTG AA -            #AACCTGCC    120                                                                 - - GGGGCCACAC CCTGGAGCGG TCTGTGTTCA GCGAGCGGAC GGAGGAGTCT TC -            #TGCCGTGC    180                                                                 - - AGTACTTCCA GTTTTATGGC TACCTGTCCC AGCAGCAGAA CATGATGCAG GA -            #CTACGTGC    240                                                                 - - GGACANGCAC CTTACCAG             - #                  - #                      - # 258                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 23:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 631 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRAITUT21                                                        (B) CLONE: 2525071F6                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #23:                          - - CTTCTACAAC ATCCTGAAAA CCTGCCGGGG CCACACCCTG GAGCGGTCTG TG -             #TTCAGCGA     60                                                                 - - GCGGACGGAG GAGTCTTCTG CCGTGCAGTA CTTCCAGTTT TATGGCTACC TG -            #TCCCAGCA    120                                                                 - - GCAGAACATG ATGCAGGACT ACGTGCGGAC AGGCACCTAC CAGCGCGGCA TC -            #CTGCAAAA    180                                                                 - - CCACACCGAC TTCAAGGACA AGATCGTTCT TGATGTTGGC TGTGGCTCTG GG -            #ATCCTGTC    240                                                                 - - GTTTTTTGCC GCCCAAGCTG GAGCACGGAA AATCTACGCG GTGGAGGCCA GC -            #ACCATGGG    300                                                                 - - CCCAGCACGC TGAGGTCTTG GTGAAGAGTA ACAACCTGAC GGGACCGCAT CG -            #TGGTCATC    360                                                                 - - CCGGGGCAAA AGTNGAAGGA AGGTGTCACT TCCCCCGAGC AAGGTGGACA TC -            #ATTAATCT    420                                                                 - - TGGGANGCCC CATGGGGCNT AANATGGNTC TTTCAAACGA AGCGGCATTG NT -            #GGGAAGAA    480                                                                 - - GCTAACCTTC CANGGGCCAA AGAAAGGTAA CTTGAAAAGN CCCCAANCCG GG -            #AAAAAAAA    540                                                                 - - TGGTTTTTCC CTAAACCATT TTGGGNGGAA NGTTCCCAAC NTTTGGGAAA CC -            #CCTTTCAA    600                                                                 - - GNGGGTTGGA AAAANGTTTC TTAAAANTNG G        - #                  - #             631                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 24:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 621 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: N/A                                                              (B) CLONE: SAEA10009P1                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #24:                          - - ATTTCNGNCN TGTTGCANAA ATGCNNAATT AGGACNCGGA CCCCAACGTC NT -             #CATCCCGG     60                                                                 - - GCAAGGTGGA GGAGGTGTCA CTCCCCGAGC AGGTGGACAT CATCATCTCG GA -            #GCCCATGG    120                                                                 - - GCTACATGCT CTTCAACGAG CGCATGCTGG ANAGCTACCT CCACGCCAAG AA -            #GTACCTGA    180                                                                 - - AGCCCAGCGG AAACATGTTT CCTACCATTG GTGACGTCCA CCTTGCACCC TT -            #CACGGATG    240                                                                 - - AACAGCTCTA CATGGAGCAG TTCACCAAGG CCAACTTCTG GTACCAGCCA TC -            #TTTCCATG    300                                                                 - - GAGTGGACCT GTCGGCCCTC CGAGGTGCCG CNNTGNNTTN TTNTTTCCGG CA -            #GCCTGTGG    360                                                                 - - TGGACACATT TGACATCCGG ATCCTGATGG CCAAGTCTGT CAAGTACACG GT -            #GAACTTCT    420                                                                 - - TAGAAGCCAA AGAAGGAGAT TTGCACAGGA TANAAATCCC ATTCAAATTC CA -            #CATGCTGC    480                                                                 - - ATTCAGGGCT GGTCCACGGC CTGGCTTTCT GGTTTGACGT TGCTTTCATC GG -            #CTCCATAA    540                                                                 - - TGACCGTGTG GCTGTCCACA GCCCCGACAG AACCCCTGAC CCACTGGTAC CA -            #GGTGCGGT    600                                                                 - - GCCTGTTCCA GTCACCACTG T           - #                  - #                     621                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 25:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 549 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: N/A                                                              (B) CLONE: SAEA03283F1                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #25:                          - - GGCTGGCTGT TGACTGCATA GTGGGCACTC GTGCTGCCAC CACCACTGCC CT -             #GGGCGCCG     60                                                                 - - GAGGACCCTT GGACAATCCC CGTGCTCATT ATGGAGCCCA TCCGGGAGTG GG -            #TGTGATTG    120                                                                 - - ACAATCCCCG TGTTGGCTAA AGGAATCAGG TTGTTGTGGC CCACGCTGGA GC -            #CACTGGCA    180                                                                 - - ATAACACTGC TCAAGTCATA GGCGGTCGGC ATCCCTGCCA CGGCCATCCC GC -            #TGCTGAGG    240                                                                 - - TTGTAGGTGC TGCCCGTGTT CCACATGTTT TCCGAGGGAG ATGTGTAGTG GG -            #AGCCGGGT    300                                                                 - - GGGGGTGAGG GCGTTGTGCC CGTGTATCTA AAGAAGGGGT TTTTCAGATC CA -            #GGAGGTTA    360                                                                 - - CTGGACTTGG AGCCGGTCTG GTCCACCTGG GCCACAATAC TGATGTCGTA GC -            #TCTGTCTT    420                                                                 - - TTGTTGGCAA TAAGCAGACA TGTCCCTGAG AGCGTGTCCC CTGCCTTGGC GA -            #ACAGTGGT    480                                                                 - - GACTGGAACA GGCACCGCAC CTGGTACCAG TGGGTCAGGG GCTCTGTCGG GG -            #CTGTGGAC    540                                                                 - - AGCCACACG                - #                  - #                      - #        549                                                                  - -  - - (2) INFORMATION FOR SEQ ID NO: 26:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 647 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: N/A                                                              (B) CLONE: SAEA01931R1                                               - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #26:                          - - GGTGGACCAG ACCGGCTCCA AGTCCAGTAA CCTCCTGGAT CTGAAAAACC CC -             #TTCTTTAG     60                                                                 - - ATACACGGGC ACAACGCCCT CACCCCCACC CGGCTCCCAC TACACATCTC CC -            #TCGGAAAA    120                                                                 - - CATGTGGAAC ACGGGCAGCA CCTACAACCT CAGCAGCGGG ATGGCCGTGG CA -            #GGGATGCC    180                                                                 - - GACCGCCTAT GACTTGAGCA GTGTTATTGC CAGTGGCTCC AGCGTGGGCC AC -            #AACAACCT    240                                                                 - - GATTCCTTTA GCCAACACGG GGATTGTCAA TCACACCCAC TCCCGGATGG GC -            #TCCATAAT    300                                                                 - - GAGCACGGGG ATTGTCCAAG GGTCCTCCGG CGCCCAGGGC AGTGGTGGTG GC -            #AGCACGAG    360                                                                 - - TGCCCACTAT GCAGTCAACA GCCAGTTCAC CATGGGCGGC CCCGCCAATC TC -            #CATGGCGT    420                                                                 - - CGCCCATGTC CATCCCGACC AACACCATGC ACTACGGGAG CTAGGGGCCC GC -            #CCCGCGGA    480                                                                 - - ACTGACAGCA CCAGGAAACC AAATGATGTC CCTGCNCGCC GCNCCCGCCG GG -            #CGGCTTTT    540                                                                 - - CCCCCTTGTA CTGGAGAAGC TCGAAACAAC CCGGTCACAG CTCTCTTTGC TA -            #TGGGAACT    600                                                                 - - GGGACATTTT TTTACACGAT GTTGCCGCCG TCCCCAAAAC GCGGGCG   - #                   647                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 27:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 655 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: LUNGFET03                                                        (B) CLONE: 1253024T6                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #27:                          - - CTGCCATATT TTACACAAAA TCATGTTGTG GGAGCCCTCG NCCCCCCTCC TG -             #CCCGCTCT     60                                                                 - - ACCCTGACCT GGGCTTGTCA TCTGCTGGAA CAGGCGCCAT GGGGCCTGCC AG -            #CCCTGCCT    120                                                                 - - GCCAGGTCCC TTAGCACCTG TCCCCCNGCC NGTCTCCAGT GGGAAGGTAG CC -            #TGGCCAGG    180                                                                 - - CGGGGCCTCC CCTTCGACGA CCAGGCCTCG GTCACAACGG ACGTGACATG CT -            #GCTTTTTT    240                                                                 - - TAATTTTATT TTTTTATGAA AAGAACCCAG TGTCAATCCG CAGACCCTCT GT -            #GAAGCCAG    300                                                                 - - GCCGGCCGGG CCGANCAAGA GGNCCTTTTC CCTAGACTCA GAGCCNCCCC GG -            #GGAAGGGG    360                                                                 - - TTTCCCCGCC GAAGGTTCAG GGNNGCCCCC TTCCCNACCA AAANGGGTTT AA -            #CCTCAAAA    420                                                                 - - TTNNAAANGN AANATCTTAC CCCCCATTNN TGGGGAAAGG GCTNCCGNTC CT -            #TNNGCCCC    480                                                                 - - NGNNTTTTTT GGNNNNNTNN TTTTTTTCCN NAANCCCCNG GAAGNTCCCN NN -            #NTTTTTNT    540                                                                 - - TTNNNNANTT AANNNTTTAN ANNNNGGNNG GNNAAAGGNN TTTNGGCCCC CN -            #TGGGGNAA    600                                                                 - - GNNNNTTGNG NGGCNAATTT NGGGGGNNAA AAANGNNNCC NNNAANGGNT TT - #TTT             655                                                                       - -  - - (2) INFORMATION FOR SEQ ID NO: 28:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 529 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: BRSTNOT09                                                        (B) CLONE: 1664573F6                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #28:                          - - TGCCTGTCTC CAGTGGGAAG GTAGCCTGGC CAGGCGGGGC CTCCCCTTCG AC -             #GACCAGGC     60                                                                 - - CTCGGTCACA ACGGACGTGA CATGCTGCTT TTTTTAATTT TATTTTTTTA TG -            #AAAAGAAC    120                                                                 - - CAGTGTCAAT CCGCAGACCC TCTGTGAAGC CAGGCCGGCC GGGCCGAGCC AG -            #CNGCCCCT    180                                                                 - - CTCCCTAGAC TCAGAGGCGC CGCGGGGAGG GGTNNCCCCG CCGAGGCTTC AG -            #GGNNNCCC    240                                                                 - - TCCCCACCAA AGGGTTCACC TCACACTTGA ATGTACANCC CANCCCACTG TC -            #GGGAAGGC    300                                                                 - - TCCGTCCTCN NCCCCTGCCT CTTGCTGCTG TCCTGTCCCC GANCCCCTGC AG -            #TCNNCTNC    360                                                                 - - NTTTNNCANT NAAGANTAGA GNAGTGGTGN NGCTTGGGCC GGAGGAAGGC AT -            #GCGGCCAN    420                                                                 - - TGGGANAANA GACACTCAAG ATTGTAGGAG GGTCTTTCCT TGAGTAAGTA GC -            #TGAGAGTC    480                                                                 - - CCTCATCTGN TAGTCAGTCT ATATGGAGGA TTCATCCTCC TGCGGAAGA  - #                  529                                                                        - -  - - (2) INFORMATION FOR SEQ ID NO: 29:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 220 base - #pairs                                                 (B) TYPE: nucleic acid                                                        (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: LUNGTUT03                                                        (B) CLONE: 1474156T1                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #29:                          - - GNNCCTTAAT TTTATTTTTT TATGAAAAGA ACCAGTGTCA ATCCGCAGAC CC -             #TCTGTGAA     60                                                                 - - GCCAGGCCGG CCGGGCCGAG CCAGCAGCCC CTCTCCCTAG ACTCAGAGGC GC -            #CGCGGGGA    120                                                                 - - GGGGTGGCCC CGCCGAGGCT TCAGGGGCCC CCTCCCCACC AAAGGGTTCA CC -            #TCACACTT    180                                                                 - - GAATGTACAA CCCACCCCAC TGTCGGGAAG GCCTCCGTCC     - #                      - #   220                                                                     - -  - - (2) INFORMATION FOR SEQ ID NO: 30:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 422 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: GI 758591                                                 - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #30:                          - - Met Ala Lys Gln Leu Gln Ala Arg Arg Leu As - #p Gly Ile Asp Tyr                            - #5                  - #10                  - #15           - - Asn Pro Trp Val Glu Phe Val Lys Leu Ala Se - #r Glu His Asp Val                           20 - #                 25 - #                 30              - - Val Asn Leu Gly Gln Gly Phe Pro Asp Phe Pr - #o Pro Pro Asp Phe                           35 - #                 40 - #                 45              - - Ala Val Glu Ala Phe Gln His Ala Val Ser Gl - #y Asp Phe Met Leu                           50 - #                 55 - #                 60              - - Asn Gln Tyr Thr Lys Thr Phe Gly Tyr Pro Pr - #o Leu Thr Lys Ile                           65 - #                 70 - #                 75              - - Leu Ala Ser Phe Phe Gly Glu Leu Leu Gly Gl - #n Glu Ile Asp Pro                           80 - #                 85 - #                 90              - - Leu Arg Asn Val Leu Val Thr Val Gly Gly Ty - #r Gly Ala Leu Phe                           95 - #                100 - #                105              - - Thr Ala Phe Gln Ala Leu Val Asp Glu Gly As - #p Glu Val Ile Ile                          110  - #               115  - #               120              - - Ile Glu Pro Phe Phe Asp Cys Tyr Glu Pro Me - #t Thr Met Met Ala                          125  - #               130  - #               135              - - Gly Gly Arg Pro Val Phe Val Ser Leu Lys Pr - #o Gly Pro Ile Gln                          140  - #               145  - #               150              - - Asn Gly Glu Leu Gly Ser Ser Ser Asn Trp Gl - #n Leu Asp Pro Met                          155  - #               160  - #               165              - - Glu Leu Ala Gly Lys Phe Thr Ser Arg Thr Ly - #s Ala Leu Val Leu                          170  - #               175  - #               180              - - Asn Thr Pro Asn Asn Pro Leu Gly Lys Val Ph - #e Ser Arg Glu Glu                          185  - #               190  - #               195              - - Leu Glu Leu Val Ala Ser Leu Cys Gln Gln Hi - #s Asp Val Val Cys                          200  - #               205  - #               210              - - Ile Thr Asp Glu Val Tyr Gln Trp Met Val Ty - #r Asp Gly His Gln                          215  - #               220  - #               225              - - His Ile Ser Ile Ala Ser Leu Pro Gly Met Tr - #p Glu Arg Thr Leu                          230  - #               235  - #               240              - - Thr Ile Gly Ser Ala Gly Lys Thr Phe Ser Al - #a Thr Gly Trp Lys                          245  - #               250  - #               255              - - Val Gly Trp Val Leu Gly Pro Asp His Ile Me - #t Lys His Leu Arg                          260  - #               265  - #               270              - - Thr Val His Gln Asn Ser Val Phe His Cys Pr - #o Thr Gln Ser Gln                          275  - #               280  - #               285              - - Ala Ala Val Ala Glu Ser Phe Glu Arg Glu Gl - #n Leu Leu Phe Arg                          290  - #               295  - #               300              - - Gln Pro Ser Ser Tyr Phe Val Gln Phe Pro Gl - #n Ala Met Gln Arg                          305  - #               310  - #               315              - - Cys Arg Asp His Met Ile Arg Ser Leu Gln Se - #r Val Gly Leu Lys                          320  - #               325  - #               330              - - Pro Ile Ile Pro Gln Gly Ser Tyr Phe Leu Il - #e Thr Asp Ile Ser                          335  - #               340  - #               345              - - Asp Phe Lys Arg Lys Met Pro Asp Leu Pro Gl - #y Ala Val Asp Glu                          350  - #               355  - #               360              - - Pro Tyr Asp Arg Arg Phe Val Lys Trp Met Il - #e Lys Asn Lys Gly                          365  - #               370  - #               375              - - Leu Val Ala Ile Pro Val Ser Ile Phe Tyr Se - #r Val Pro His Gln                          380  - #               385  - #               390              - - Lys His Phe Asp His Tyr Ile Arg Phe Cys Ph - #e Val Lys Asp Glu                          395  - #               400  - #               405              - - Ala Thr Leu Gln Ala Met Asp Glu Lys Leu Ar - #g Lys Trp Lys Val                          410  - #               415  - #               420              - - Glu Leu                                                                   - -  - - (2) INFORMATION FOR SEQ ID NO: 31:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 425 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: GI 1050752                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #31:                          - - Met Asn Tyr Ser Arg Phe Leu Thr Ala Thr Se - #r Leu Ala Arg Lys                            - #5                  - #10                  - #15           - - Thr Ser Pro Ile Arg Ala Thr Val Glu Ile Me - #t Ser Arg Ala Pro                           20 - #                 25 - #                 30              - - Lys Asp Ile Ile Ser Leu Ala Pro Gly Ser Pr - #o Asn Pro Lys Val                           35 - #                 40 - #                 45              - - Phe Pro Phe Lys Ser Ala Val Phe Thr Val Gl - #u Asn Gly Ser Thr                           50 - #                 55 - #                 60              - - Ile Arg Phe Glu Gly Glu Met Phe Gln Arg Al - #a Leu Gln Tyr Ser                           65 - #                 70 - #                 75              - - Ser Ser Tyr Gly Ile Pro Glu Leu Leu Ser Tr - #p Leu Lys Gln Leu                           80 - #                 85 - #                 90              - - Gln Ile Lys Leu His Asn Pro Pro Thr Val As - #n Tyr Ser Pro Asn                           95 - #                100 - #                105              - - Glu Gly Gln Met Asp Leu Cys Ile Thr Ser Gl - #y Cys Gln Asp Gly                          110  - #               115  - #               120              - - Leu Cys Lys Val Phe Glu Met Leu Ile Asn Pr - #o Gly Asp Thr Val                          125  - #               130  - #               135              - - Leu Val Asn Glu Pro Leu Tyr Ser Gly Ala Le - #u Phe Ala Met Lys                          140  - #               145  - #               150              - - Pro Leu Gly Cys Asn Phe Ile Ser Val Pro Se - #r Asp Asp Cys Gly                          155  - #               160  - #               165              - - Ile Ile Pro Glu Gly Leu Lys Lys Val Leu Se - #r Gln Trp Lys Pro                          170  - #               175  - #               180              - - Glu Asp Ser Lys Asp Pro Thr Lys Arg Thr Pr - #o Lys Phe Leu Tyr                          185  - #               190  - #               195              - - Thr Ile Pro Asn Gly Asn Asn Pro Thr Gly As - #n Ser Leu Thr Gly                          200  - #               205  - #               210              - - Asp Arg Lys Lys Glu Ile Tyr Glu Leu Ala Ar - #g Lys Tyr Asp Phe                          215  - #               220  - #               225              - - Leu Ile Ile Glu Asp Asp Pro Tyr Tyr Phe Le - #u Gln Phe Thr Lys                          230  - #               235  - #               240              - - Pro Trp Glu Pro Thr Phe Leu Ser Met Asp Va - #l Asp Gly Arg Val                          245  - #               250  - #               255              - - Ile Arg Ala Asp Ser Leu Ser Lys Val Ile Se - #r Ser Gly Leu Arg                          260  - #               265  - #               270              - - Val Gly Phe Ile Thr Gly Pro Lys Ser Leu Il - #e Gln Arg Ile Val                          275  - #               280  - #               285              - - Leu His Thr Gln Ile Ser Ser Leu His Pro Cy - #s Thr Leu Ser Gln                          290  - #               295  - #               300              - - Leu Met Ile Ser Glu Leu Leu Tyr Gln Trp Gl - #y Glu Glu Gly Phe                          305  - #               310  - #               315              - - Leu Ala His Val Asp Arg Ala Ile Asp Phe Ty - #r Lys Asn Gln Arg                          320  - #               325  - #               330              - - Asp Phe Ile Leu Ala Ala Ala Asp Lys Trp Le - #u Arg Gly Leu Ala                          335  - #               340  - #               345              - - Glu Trp His Val Pro Lys Ala Gly Met Phe Le - #u Trp Ile Lys Val                          350  - #               355  - #               360              - - Asn Gly Ile Ser Asp Ala Lys Lys Leu Ile Gl - #u Glu Lys Ala Ile                          365  - #               370  - #               375              - - Glu Arg Glu Ile Leu Leu Val Pro Gly Asn Se - #r Phe Phe Val Asp                          380  - #               385  - #               390              - - Asn Ser Ala Pro Ser Ser Phe Phe Arg Ala Se - #r Phe Ser Gln Val                          395  - #               400  - #               405              - - Thr Pro Ala Gln Met Asp Leu Val Phe Gln Ar - #g Leu Ala Gln Leu                          410  - #               415  - #               420              - - Ile Lys Asp Val Ser                                                                      425                                                            - -  - - (2) INFORMATION FOR SEQ ID NO: 32:                                   - -      (i) SEQUENCE CHARACTERISTICS:                                                 (A) LENGTH: 343 amino - #acids                                                (B) TYPE: amino acid                                                          (C) STRANDEDNESS: single                                                      (D) TOPOLOGY: linear                                                 - -    (vii) IMMEDIATE SOURCE:                                                         (A) LIBRARY: GenBank                                                          (B) CLONE: GI 1808648                                                - -     (xi) SEQUENCE DESCRIPTION: SEQ ID NO: - #32:                          - - Met Glu Val Ser Cys Gly Gln Ala Glu Ser Se - #r Glu Lys Pro Asn                            - #5                  - #10                  - #15           - - Ala Glu Asp Met Thr Ser Lys Asp Tyr Tyr Ph - #e Asp Ser Tyr Ala                           20 - #                 25 - #                 30              - - His Phe Gly Ile His Glu Glu Met Leu Lys As - #p Glu Val Arg Thr                           35 - #                 40 - #                 45              - - Leu Thr Tyr Arg Asn Ser Met Phe His Asn Ar - #g His Leu Phe Lys                           50 - #                 55 - #                 60              - - Asp Lys Val Val Leu Asp Val Gly Ser Gly Th - #r Gly Ile Leu Cys                           65 - #                 70 - #                 75              - - Met Phe Ala Ala Lys Ala Gly Ala Arg Lys Va - #l Ile Gly Ile Val                           80 - #                 85 - #                 90              - - Cys Ser Ser Ile Ser Asp Tyr Ala Val Lys Il - #e Val Lys Ala Asn                           95 - #                100 - #                105              - - Lys Leu Asp His Val Val Thr Ile Ile Lys Gl - #y Lys Val Glu Glu                          110  - #               115  - #               120              - - Val Glu Leu Pro Val Glu Lys Val Asp Ile Il - #e Ile Ser Glu Trp                          125  - #               130  - #               135              - - Met Gly Tyr Cys Leu Phe Tyr Glu Ser Met Le - #u Asn Thr Val Leu                          140  - #               145  - #               150              - - Tyr Ala Arg Asp Lys Trp Leu Ala Pro Asp Gl - #y Leu Ile Phe Pro                          155  - #               160  - #               165              - - Asp Arg Ala Thr Leu Tyr Val Thr Ala Ile Gl - #u Asp Arg Gln Tyr                          170  - #               175  - #               180              - - Lys Asp Tyr Lys Ile His Trp Trp Glu Asn Va - #l Tyr Gly Phe Asp                          185  - #               190  - #               195              - - Met Ser Cys Ile Lys Asp Val Ala Ile Lys Gl - #u Pro Leu Val Asp                          200  - #               205  - #               210              - - Val Val Asp Pro Lys Gln Leu Val Thr Asn Al - #a Cys Leu Ile Lys                          215  - #               220  - #               225              - - Glu Val Asp Ile Tyr Thr Val Lys Val Glu As - #p Leu Thr Phe Thr                          230  - #               235  - #               240              - - Ser Pro Phe Cys Leu Gln Val Lys Arg Asn As - #p Tyr Val His Ala                          245  - #               250  - #               255              - - Leu Val Ala Tyr Phe Asn Ile Glu Phe Thr Ar - #g Cys His Lys Arg                          260  - #               265  - #               270              - - Thr Gly Phe Ser Thr Ser Pro Glu Ser Pro Ty - #r Thr His Trp Lys                          275  - #               280  - #               285              - - Gln Thr Val Phe Tyr Met Glu Asp Tyr Leu Th - #r Val Lys Thr Gly                          290  - #               295  - #               300              - - Glu Glu Ile Phe Gly Thr Ile Gly Met Arg Pr - #o Asn Ala Lys Asn                          305  - #               310  - #               315              - - Asn Arg Asp Leu Asp Phe Thr Ile Asp Leu As - #p Phe Lys Gly Gln                          320  - #               325  - #               330              - - Leu Cys Glu Leu Ser Cys Ser Thr Asp Tyr Ar - #g Met Arg                                  335  - #               340                                   __________________________________________________________________________

What is claimed is:
 1. An isolated and purified polynucleotide encodinga polypeptide comprising an amino acid sequence selected from the groupconsisting of SEQ ID NO: 1, SEQ ID NO:2, and SEQ ID NO:3.
 2. An isolatedand purified polynucleotide which hybridizes under hybridizationconditions of 250 mM NaCl, 25 mM trisodium citrate, 1% SDS, 50%formamide, and 200 μg/ml ssDNA at 42° C., and wash conditions of 15 mMNaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68° C. to thepolynucleotide of claim
 1. 3. An isolated and purified polynucleotidewhich is completely complementary to the polynucleotide of claim
 1. 4.An isolated and purified polynucleotide selected from the groupconsisting of SEQ ID NO:4, SEQ ID NO:5, and SEQ ID NO:6.
 5. An isolatedand purified polynucleotide which is completely complementary to thepolynucleotide of claim
 4. 6. An expression vector comprising thepolynucleotide of claim
 1. 7. A host cell comprising the expressionvector of claim
 6. 8. A method for producing a polypeptide comprisingthe amino acid sequence selected from the group consisting of SEQ IDNO:1, SEQ ID NO:2, and SEQ ID NO:3, the method comprising the stepsof:(a) culturing the host cell of claim 7 under conditions suitable forthe expression of the polypeptide; and (b) recovering the polypeptidefrom the host cell culture.
 9. A method for detecting a polynucleotideencoding a polypeptide comprising the amino acid sequence selected fromthe group consisting of SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, in abiological sample containing nucleic acids, the method comprising thesteps of:(a) hybridizing the polynucleotide of claim 3 underhybridization conditions of 250 mM NaCl, 25 mM trisodium citrate, 1%SDS, 50% formamide, and 200 μg/ml ssDNA at 42° C., to at least one ofthe nucleic acids in the biological sample, thereby forming ahybridization complex; (b) subjecting the hybridization complex to washconditions of 15 mM NaCl, 1.5 mM trisodium citrate, and 0.1% SDS at 68°C.; and (c) detecting the hybridization complex, wherein the presence ofthe hybridization complex correlates with the presence of thepolynucleotide encoding the polypeptide in the biological sample. 10.The method of claim 9 further comprising amplifying the nucleic acids ofthe biological sample by the polymerase chain reaction prior to thehybridizing step.